Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation

Chen, John J.; Nathaniel, Diane; Raghavan, Preethi; Nelson, Maxine; Tian, Ruilin; Tse, Eric; Hong, Jin-Hyuk; See, Stephanie K.; Mok, Sue-Ann; Hein, Marco Y.; Southworth, Daniel R.; Grinberg, Lea T.; Gestwicki, Jason E.; Leonetti, Manuel D.; Kampmann, Martin

doi:10.1074/jbc.ra119.009432

Cited by 129 publications

(108 citation statements)

References 70 publications

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“…Following established protocols for validation of primary screens, we targeted each gene with two distinct sgRNAs. 20–22 We found that depletion of 6/7 (86 %) of the hits identified from the “high”/”low” and 1/2 (50 %) of the hits from the “medium”/”low” gave rise to phenotypes consistent with the pooled screen result (Fig. 3b, S3b-c).…”

Section: Resultssupporting

confidence: 78%

A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling

Semesta

Tian

Kampmann

et al. 2020

Preprint

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G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and weaker phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with weaker phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.Author summaryCells sense and respond to changes in their surrounding environment through G protein-coupled receptors (GPCRs) and their associated cascades. The proper function of these pathways is essential to human physiology, and GPCRs have become a prime target for drug development for a range of human diseases. Therefore, it is of utmost importance to be able to map how these pathways operate to enable cells to fine-tune their responsiveness. Here, we describe a screening approach that we have devised to systematically identify regulators of GPCR function. We have developed a sortable reporter system and coupled that with silencing of genes across the entire human genome in order to uncover a range of novel mediators of GPCR activity. We characterize a few of these new regulators and show that they function at different steps of the cascade. Therefore, this study serves as proof of principle for the new screening platform. We envision that the approach can be used to dissect additional dimensions of GPCR function, including regulators of drug-specific responses, functional characterization of receptor features, and identification of novel drugs, and thus advance a genome-scale understanding of these critical pathways.

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Section: Resultssupporting

confidence: 78%

A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling

Semesta

Tian

Kampmann

et al. 2020

Preprint

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“…To learn more about the steps between uptake of tau seeds and subsequent aggregation, Chen et al use their modified FRETbased tau aggregation assay in a CRISPRi-based screen to identify regulators of tau uptake and propagation. They first confirmed that they could observe a prion-like propagation of tau aggregation within the cell using seed material of recombinant aggregated tau or aggregates purified from AD patient brains (7). As in previous studies, the authors observed tau uptake into the cell, presumably via endocytosis, followed by release into the cytosol, where propagation of tau aggregation occurs.…”

supporting

confidence: 68%

Leaky endosomes push tau over the seed limit

Ugbode

Fort-Aznar

Sweeney

2019

Journal of Biological Chemistry

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“…The strict requirement for Draper in ORN-to-glia and ORN-to-glia-to-PN aggregate transfer indicates that neuronal mHtt ex1 and glial wtHtt ex1 proteins physically interact during or following phagocytic engulfment, requiring breaching of one or more membrane barriers that normally exclude phagocytosed material from the glial cytoplasm. In support of this, prion-like aggregates formed by α-synuclein, tau, and mHtt ex1 have the capacity to rupture cell surface or endolysosomal membranes to directly access the cytoplasmic compartment (Chen et al, 2019; Falcon et al, 2018; Flavin et al, 2017; Zeineddine et al, 2015). It is also possible that phagocytosis becomes dysregulated in diseased or aged brains.…”

Section: Discussionmentioning

confidence: 90%

“…Accumulating evidence supports the idea that intracellular aggregates formed by tau, α-synuclein, TDP-43, SOD1, and mutant huntingtin (mHtt) transfer from cell-to-cell and self-replicate by recruiting natively-folded versions of the same protein, analogous to how infectious prion protein (PrP Sc ) templates the conformational change of soluble PrP C in prion diseases (Vaquer-Alicea and Diamond, 2019). Numerous studies have pointed to roles for endocytosis and exocytosis (Asai et al, 2015; Babcock and Ganetzky, 2015; Chen et al, 2019; Holmes et al, 2013; Lee et al, 2010; Zeineddine et al, 2015), membrane permeabilization (Chen et al, 2019; Falcon et al, 2018; Flavin et al, 2017; Zeineddine et al, 2015), tunneling nanotubes (Costanzo et al, 2013; Sharma and Subramaniam, 2019), and neuronal activity (Wu et al, 2016) in entry and/or exit of pathogenic protein assemblies from cells, but the exact mechanisms by which amyloid aggregates or on-pathway intermediates could cross one or more biological membranes in the highly complex central nervous system (CNS) remain an enigma.…”

Section: Introductionmentioning

confidence: 99%

Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

Donnelly

DeLorenzo

Zaya

et al. 2019

Preprint

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14Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with 15 many neurodegenerative diseases spread from cell to cell through the brain in a manner akin to 16 infectious prions. Here, we show that mutant huntingtin (mHtt) aggregates associated with Huntington 17 disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila 18 olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal 19 activity and blocked by caspase inhibition in presynaptic neurons, implicating synaptic dysfunction and 20 cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires 21 the scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that 22 phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected 23 neurons. These findings expand our understanding of phagocytic glia as double-edged players in 24 neurodegeneration-they clear neurotoxic protein aggregates, but also provide opportunities for prion-25 like seeds to evade phagolysosomal degradation and propagate further in the brain. 26 93 5 RESULTS 94Anterograde transfer of prion-like mHtt aggregates across synapses in the adult fly olfactory system 95 Aggregates formed by N-terminal fragments of mHtt generated by aberrant splicing (e.g., exon 96 1; Httex1) (Sathasivam et al., 2013) or caspase cleavage (e.g., exon 1-12; Httex1-12) (Graham et al., 2006) 97 (Fig. 1A) accumulate in HD patient brains, are highly cytotoxic, and spread between cells in culture and 98 in vivo (Babcock and Ganetzky, 2015; Costanzo et al., 2013; Pearce et al., 2015; Pecho-Vrieseling et 99 al., 2014; Ren et al., 2009). We have previously established transgenic Drosophila that employ binary 100 expression systems [e.g., Gal4-UAS, QF-QUAS, or LexA-LexAop (Riabinina and Potter, 2016)] to 101 express fluorescent protein (FP) fusions of Httex1 in non-overlapping cell populations in order to monitor 102 cell-to-cell transfer of mHttex1 aggregates in intact brains (Donnelly and Pearce, 2018; Pearce et al., 103 2015). Our experimental approach (Fig. 1B) exploits the fact that wtHttex1 proteins only aggregate upon 104 encountering mHttex1 aggregate seeds (Chen et al., 2001; Preisinger et al., 1999), such that transfer of 105 mHttex1 aggregates from "donor" cells is reported by conversion of cytoplasmic wtHttex1 from its normally 106 soluble, diffuse state to a punctate, aggregated state in "acceptor" cells. To confirm that mHttex1 107 nucleates the aggregation of wtHttex1 in fly neurons, we co-expressed FP-fusions of these two proteins 108 using pan-neuronal elav[C155]-Gal4. In flies expressing only mCherry-tagged mHttex1 (Httex1Q91-109 mCherry) pan-neuronally, aggregates were visible as discrete mCherry+ puncta throughout neuronal 110 cell bodies and neuropil regions of adult fly brains ( Fig. 1-figure supplement 1A). By contrast, GFP-111 tagged wtHttex1 (Httex1Q25-GFP) was expressed di...

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Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation

Cited by 129 publications

References 70 publications

A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling

A high-throughput CRISPR interference screen for dissecting functional regulators of GPCR/cAMP signaling

Leaky endosomes push tau over the seed limit

Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses

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