Large-scale bidirectional arrayed genetic screens identify OXR1 and EMC4 as modifiers of α-synuclein aggregation

  1. Sandesh Neupane
  2. Lea Nikolić
  3. Lorenzo Maraio
  4. Thomas Goiran
  5. Nathan Karpilovsky
  6. Stefano Sellitto
  7. Vangelis Bouris
  8. Jiang-An Yin
  9. Ronald Melki
  10. Edward A. Fon
  11. Elena De Cecco
  12. Adriano Aguzzi

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Abstract

In Parkinson’s disease and other synucleinopathies, α-synuclein (α-Syn) misfolds and forms Ser 129 -phosphorylated aggregates (pSyn 129 ). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn 129 modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to α-Syn fibrils, pSyn 129 signals were recorded by high-throughput fluorescence microscopy and aggregates were identified by image analysis. We found that pSyn 129 was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn 129 was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn 129 across diverse α-Syn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in α-Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.

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    Referee #1

    1. The authors should provide more information when...

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    • Though this is not stated in the MS
    1. Figure 6: Why has only...

    Response: We expanded the comparison

    Minor comments:

    1. The text contains several...

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    Referee #2

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    Referee #3

    Evidence, reproducibility and clarity

    In this work Neupane et al used large-scale robust CRISPR-based gene activation and ablation screens to identify novel regulators of α-synuclein pathology in synucleinopathies using as read-out p-αSyn129 signals by high-throughput fluorescence microscopy. The authors reveal that mitochondrial protein OXR1 promotes Ser129-phosphorylated αSyn aggregation, while ER-associated EMC4 suppresses it via enhanced autophagic clearance, highlighting new possible mechanistic pathways in disease progression of alpha-synucleinopathies.

    Major comments:

    1. As correctly pointed out by the authors in Introduction p-Syn is associated with aggregates, but its functional role is far to be clear and both neuroprotective or pro-aggregations effects have been proposed. Further it has been shown that, physiological neuronal activity augments Ser129-phospho αSyn, which is a trigger for protein-protein interactions, which in turn is necessary for mediating αSyn function at the synapse (https://doi.org/10.1016/j.neuron.2023.11.020). As a consequence modulation of p- p-αSyn as possible therapeutical target for PD and synucleinopathies is quite a complicate matter. The assumption, on which the whole paper is based, that increase in p-αSyn equates to αSyn aggregation and disease progression is rather weak to this reviewer, unless further validation of it is provided. Indeed while the authors performed experiments on human iPSC-derived cortical and dopaminergic neurons on p-αSyn analysis, any measurement of αSyn aggregates/oligomers, and neuronal degeneration is provided. It is recommended to provid this experiments ideally using different tecnique like αSyn-Proximity Ligation Assay for measurements of oligomers, as it has been largely validated in autoptic brains of PD, MSA and DLB patients (doi: 10.1007/s00401-025-02871-w.), as well as cell viability/apoptosis and neurites degeneration measurements upon OXR1 and EMC4 modulation in iPSC derived cortical and dopaminergic neurons.
    2. The authors claims in Results page 5: "The absence of cytoplasmic pSyn129 signal in HEK293 cells lacking α-Syn overexpression demonstrates that elevated α-Syn levels are essential to drive robust and rapid aggregation. Moreover, it indicates that the 81A antibody selectively recognizes de novo aggregates rather than the recombinant seeds". The fact that ab81A recognize deNovo aggregates and not rec seeds is quite speculative, not supported by data, and might rather indicate that ab 81A does not recognize aggregates. Thus this further implays that other technology like for example Seeding amplification assays are being employed by the authors in addition to p-αSyn129 signals in validation experiments for example in genetic PD (ideally GBA1 or LRRK2) IPSC-derived dopaminergic neurons.

    Minor comments:

    1. The strain-specific effects especially from patients-derived fibrils of OXR1 activation and EMC4 depletion on pSyn levels is rather weak in comparison with RAB3 and PIKFYVE (fig 3F-G) and therefore the expected relevance of these results especially in vivo in patients should be better clarified and modulated in discussion
    2. In discussion authors write "We observed that OXR1 activation preferentially increases α-Syn aggregates phosphorylation (EP1536Y) in neuronal somata, suggesting that mitochondrial dysfunction exacerbates α-Syn phosphorylation in later-stage aggregates." This is quite a surprising result since distal axonal endings are particularly susceptible to mitochondrial impairments for anatomical and physiologically reasons and if p-αSyn129 accumulation is driven by mithocondrial disfunction as suggested by this paper, this should be detected in neurites as well. Please clarify.
    3. Authors say that they targeted mitochondrial, trafficking, and motility (MTM) genes in human cellular models. While mitochondrial and trafficking is clear in the context of Parkinson and neurodegnerative disease, less clear is the motility genes. Please expand on this.

    Significance

    This is a well written, comprehensive study with a well characterized, robust CRISPR-based gene activation and ablation screening pipeline to identify novel regulators of α-synuclein pathology. Methodology is rigorous and clearly described and results are well presented. The major limitation relays in the validation experiments where only one main read-out that is p-αSyn129 fluorescence signal is employed, limiting the significance and impact of the presented results. I believe that the basic science community might benefit principally of the proposed methodology of a large high-throughput screening to modulate a large set of genes, a platform that in principle might be used also for other scientific questions.

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    Referee #2

    Evidence, reproducibility and clarity

    In the present study, Neupane et al. performed arrayed CRISPR activation and ablation screens, targeting genes related to mitochondria, trafficking and motility, to identify genes that modulate the presence of Ser 129 phosphorylated alpha-synuclein aggregates (pSyn129) upon administration of exogenous preformed alpha-synuclein fibrils. The screens have been performed in HEK cells stably overexpressing alpha-synuclein in two independent replicates, and hits have been further validated in induced pluripotent stem cell derived forebrain and dopaminergic neurons. Following functional validations, the authors conclude that enhancing the expression of OXR1 results in a modest increase in the number of pSyn129 puncta within cells, and their size, while partial loss of EMC4 expression reduces these puncta. To date some pre-print studies have used genome-wide CRISPR screening to identify modifiers of the accumulation of alpha-synuclein preformed fibrils in cells, suggesting the importance of uptake and endolysosomal trafficking for the propagation of alpha-synuclein aggregates in recipient cells. Although the topic is of interest in the field of Parkinson's disease and synucleinopathies in general, the readout of the present screen (presence of pSyn129) is not very sensitive and without investigating endogenous alpha-synuclein or cell homeostasis in neuronal models limits the stated conclusions.

    Major comments:

    • Please clarify whether the positive control genes RAB13 and PIKFYVE were nominated hits within the CRISPR screens. Specifically, the authors state that the positive control of the CRISPRa screen was RAB13, expected to reduce pSyn129 upon overexpression, nevertheless this gene does not appear as a hit in the CRISPRa volcano plot (although present in table S1 but not making the cutoff). In figure 2D, activation of RAB13 does not seem to impact the main readout phenotype. Moreover, in the CRISPRo screen, PIKFYVE was used, but this gene is also not presented as a hit linked to reducing pSyn129 in the CRISPRo plot. If these control genes do not come up as hits, it is difficult to support the conclusions of the screen.
    • The effect size for screen hits presented in figure 2A/B is rather small. It is difficult to interpret the power of these findings in the absence of uptake efficiency controls, such as dextrans of appropriate molecular weights.
    • The readout of the screen is not very sensitive, and it is unclear what it represents. Specifically, in Figure 2F, G the authors validate the hits OXR1 and EMC4, showing a small effect, albeit statistically significant. The authors should strengthen this data by adding more experiments addressing, for instance, what the pSyn spot area and spot intensity signify for the cell. Some experiments in a neuronal context are important, including SNCA KO as a negative control.
    • It is unclear why the authors chose to follow up on the OXR1 and EMC4 hits. Please explain the rationale for follow-up studies.
    • Generally, the notable difference in the number of pSyn129+ cells in the non-targeting across various experiments (including Fig.1G/I compared to Fig.2G/I or Fig. 3F/G or flow cytometry experiment) suggests the readout is not very sensitive.

    For instance, in figure S3 it would be important to add an experiment controlling for cell number as opposed to LDH release, as the micrographs show some differences in cells number, e.g. in the ntg vs. EMC4 condition.

    • The data is not sufficient to suggest that OXR1 and EMC4 are strong modulators of alpha-synuclein aggregation, as the authors suggest based on figures 2 and 3 that show statistically significant difference and a rather small effect size. It is important to provide more insight into how these genes may affect endogenous alpha-synuclein and cellular homeostasis in more detail, especially in neuronal models. Further investigating the hits in this direction in additional genetic backgrounds would also increase the relevance of the findings, e.g. in SNCA triplication or GBA-PD neurons.
      In Fig. S8B the immunoblot analysis shows there may be an effect of EMC4 and OXR1 CRISPRa on α-synuclein levels; please quantify for both iPSC-derived cortical neurons and dopaminergic neurons.
    • The pattern of tyrosine hydroxylase staining in Figure 5F does not seem specific or as expected for iPSC-derived dopaminergic neurons. Furthermore, since endogenous SNCA expression is expected to be analogous to the expression of TH (with TH+ cells expressing higher SNCA), it would be important to compare pSyn129 between TH+ cells and/or relative to the TH+ area.

    Minor comments:

    • The authors report that RAB13 overexpression reduces pSyn129⁺ prevalence, whereas RAB13 ablation (CRISPRo screen) enhances pSyn129⁺ levels (Figures 2D-2E). Please revise as these specific figures show no effects for this gene.
    • Please specify how many individual cells (approximately) were quantified in each figure legend.
    • Figure 3F/G may be better as a supplemental figure since it does not add to the conclusions of the study.
    • It would be good to clarify for the reader some of the genes that serve as positive controls for the screen's readout (as shown in Fig. S2D/G).
    • It would be helpful to further clarify which cell type was used in each figure legend.

    Significance

    Important topic but their experimental design limits the significance of their findings. Hard to improve the work in a reasonable amount of time. Also many technical issues.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    This study by Neupane et al. investigates modulators of α-synuclein aggregation, focusing on Ser129-phosphorylated α-synuclein (pSyn129), a pathological hallmark of Parkinson's disease (PD). The authors performed high-content image-based, arrayed CRISPR activation (CRISPRa) and knockout (CRISPRo) screens targeting > 2300 genes related to mitochondrial function, intracellular trafficking, and cytoskeletal reorganization. Using α-Syn overexpressing HEK293 cells, they identified OXR1 and EMC4 as novel modulators of pSyn129 abundance. Key findings were that activation of the mitochondrial protein OXR1 increased pSyn129 by decreasing ATP levels, while ablation of the ER-associated protein EMC4 reduced pSyn129 by enhancing autophagic flux and lysosomal clearance. These findings were validated in human iPSC-derived cortical and dopaminergic neurons.

    My major comments have to do with statistical methods and with significance of their findings.

    Major comments:

    Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them?

    The claims and conclusions are generally well-supported by the presented data. The dual CRISPRa/CRISPRo screen provides a robust initial discovery platform, and the validation in iPSC-derived neurons strengthens the findings and their translational relevance. The mechanistic insights into OXR1 (ATP levels) and EMC4 (autophagic flux, lysosomal clearance) are supported by the described experiments. The use of two antibodies (81A and EP1536Y) for pSyn129 also enhances confidence in the measurements. I had a few questions about the statistical methods. The main concern I have about methodology for the screen is whether the authors have corrected for multiple hypotheses in their discovery screen. This is not clear from the text, methods, or legends (for Figures 2A/2B/2C).

    • Figure 1B suggests a very large range of activation (multiple orders of magnitude) in the initial screen. What is the relationship between level of expression change and functional effect across the screen? How upregulated/downregulated are OXR1 and EMC4 at the mRNA and protein levels?
    • Supplemental Figure S2D: Why do the non-targeting controls differ from the majority of the CRISPRa genes? If I am reading the figure correctly, it seems strange that the vast majority of the CRISPRa gene targets reduces pSyn pathology relative to the non-targeting controls (which is why I am wondering whether the level of increased expression correlates with the level of functional effect).
    • In Figure 2A/B/C, is the p-value adjusted in any way for multiple comparisons? If so, this should be indicated in the legend. If not, why not? (The potential for false positives in a screen is very large and requires correction for multiple comparisons.)
    • Figure 3: It's interesting that different seeding materials have different effects. However, it's quite surprising that the authors find less seeding with MSA-derived material in both the CRISPRa and CRISPRo context. This contradicts the work of Peng and coauthors (PMID 29743672) who find that MSA-derived material is much more potent in seeding aggregates in a number of different cell types. Do the authors have any thoughts about why this is the case?
    • Figure 7A: pSyn129 image in the non-targeting control is poor - the very bright dots look like artifact. Not clear why the authors don't corroborate with EP1536Y antibody as they do in Figure 5.
    • Overall methodology: Are the pSyn inclusions soluble? This could be easily determined by performing 1% TritonX extraction, for example, and it helps us understand how "pathological" the inclusions are.
    • OPTIONAL: The authors perform some interesting experiments looking at genes affected downstream by, for example, OXR1 over-expression. It would be useful to understand whether the upstream effect is dependent on downstream effect. This could be tested by performing double perturbations (e.g. OXR1 overexpression and CCL8 knockout or ALDOC upregulation).
    • OPTIONAL: The link between EMC4 ablation and enhanced ER-driven autophagic flux/lysosomal clearance could be corroborated with additional experiments. E.g.: Does EMC4 normally inhibit this pathway? Or only in the context of aSyn fibril seeding?

    Are the suggested experiments realistic in terms of time and resources?

    The OPTIONAL experiments are generally feasible as they employ methods that the lab is already using in this paper.

    Are the experiments adequately replicated and statistical analysis adequate?

    See comment about multiple hypothesis testing above.

    Significance

    This is a well-designed, difficult-to-accomplish study that expands the landscape of pS129Syn modulators. The validation of the primary hits identified in HEK293 cells in iPSC-derived neurons gives the findings greater relevance.

    Strengths:

    • Novelty: Using an unbiased and high-throughput approach, the study identifies two novel regulators of α-Syn aggregation, namely OXR1 and EMC4.
    • Methodological Rigor: The use of arrayed CRISPRa/CRISPRo screens with high-content imaging is powerful and difficult to accomplish. Methodologically, this is a tour de force.
    • Orthogonal Validation: The use of multiple α-Syn fibril polymorphs/strains and different antibodies (81A, EP1536Y) strengthens the robustness of the findings.

    Limitations:

    • It's not clear to me that pSyn129 is the ultimate readout. At a minimum, we should know something about the solubility of the inclusions. Some panels (e.g. Figure 7A) are not very informative in terms of what the authors are calling pSyn129+.
    • The study relies on in vitro cellular models. While iPSC-derived neurons are relevant, the complexity of the brain environment, including glial cell interactions is not fully captured. This is fine for an initial report, but it does limit the significance.
    • OXR1 and EMC4 seem to be very generic modulators. It's not clear to me that their effects are specific to aSyn or to PD in any way - they might just be effects on very basic cellular functions that would be applicable to a number of stressors or proteinopathies. Maybe that is fine (we probably need to get rid of tau aggregates, too!), but I don't think the authors can claim that they have identified "organelle-specific genetic nodes of aSyn pathology" since they biased their screen towards mitochondria and they don't test any other pathological aggregates. Moreover, from a translational perspective, it's not clear to me that implicating the antioxidant pathway or lysosomal/autophagosomal pathways in the pathogenesis of PD is new, and it's not clear that the specific genes identified would make good therapeutic targets.
  5. Version published to 10.1101/2025.06.10.658866 on bioRxiv