Sequential CRISPR screening reveals partial NatB inhibition as a strategy to mitigate alpha-synuclein levels in human neurons.

Alpha-synuclein (αSyn) protein levels correlate with the risk and severity of Parkinson's disease and related neurodegenerative diseases. Lowering αSyn is being actively investigated as a therapeutic modality. Here, we systematically map the regulatory network that controls endogenous αSyn using sequential CRISPR-knockout and -interference screens in an αSyn gene (SNCA)-tagged cell line and induced pluripotent stem cell-derived neurons (iNeurons). We uncover αSyn modifiers at multiple regulatory layers, with amino-terminal acetyltransferase B (NatB) enzymes being the most potent endogenous αSyn modifiers in both cell lines. Amino-terminal acetylation protects the cytosolic αSyn from rapid degradation by the proteasome in a Ube2w-dependent manner. Moreover, we show that pharmacological inhibition of methionyl-aminopeptidase 2, a regulator of NatB complex formation, attenuates endogenous αSyn in iNeurons carrying SNCA triplication. Together, our study reveals several gene networks that control endogenous αSyn, identifies mechanisms mediating the degradation of nonacetylated αSyn, and illustrates potential therapeutic pathways for decreasing αSyn levels in synucleinopathies.

CRISPR screen for protein inclusion formation uncovers a role for SRRD in the regulation of intermediate filament dynamics and aggresome assembly.

The presence of large protein inclusions is a hallmark of neurodegeneration, and yet the precise molecular factors that contribute to their formation remain poorly understood. Screens using aggregation-prone proteins have commonly relied on downstream toxicity as a readout rather than the direct formation of aggregates. Here, we combined a genome-wide CRISPR knockout screen with Pulse Shape Analysis, a FACS-based method for inclusion detection, to identify direct modifiers of TDP-43 aggregation in human cells. Our screen revealed both canonical and novel proteostasis genes, and unearthed SRRD, a poorly characterized protein, as a top regulator of protein inclusion formation. APEX biotin labeling reveals that SRRD resides in proximity to proteins that are involved in the formation and breakage of disulfide bonds and to intermediate filaments, suggesting a role in regulation of the spatial dynamics of the intermediate filament network. Indeed, loss of SRRD results in aberrant intermediate filament fibrils and the impaired formation of aggresomes, including blunted vimentin cage structure, during proteotoxic stress. Interestingly, SRRD also localizes to aggresomes and unfolded proteins, and rescues proteotoxicity in yeast whereby its N-terminal low complexity domain is sufficient to induce this affect. Altogether this suggests an unanticipated and broad role for SRRD in cytoskeletal organization and cellular proteostasis.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

A subpopulation of lipogenic brown adipocytes drives thermogenic memory.

Sustained responses to transient environmental stimuli are important for survival. The mechanisms underlying long-term adaptations to temporary shifts in abiotic factors remain incompletely understood. Here, we find that transient cold exposure leads to sustained transcriptional and metabolic adaptations in brown adipose tissue, which improve thermogenic responses to secondary cold encounter. Primary thermogenic challenge triggers the delayed induction of a lipid biosynthesis programme even after cessation of the original stimulus, which protects from subsequent exposures. Single-nucleus RNA sequencing and spatial transcriptomics reveal that this response is driven by a lipogenic subpopulation of brown adipocytes localized along the perimeter of Ucp1hi adipocytes. This lipogenic programme is associated with the production of acylcarnitines, and supplementation of acylcarnitines is sufficient to recapitulate improved secondary cold responses. Overall, our data highlight the importance of heterogenous brown adipocyte populations for 'thermogenic memory', which may have therapeutic implications for leveraging short-term thermogenesis to counteract obesity.

Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics.

The field of induced proximity therapeutics is in its ascendancy but is limited by a lack of scalable tools to systematically explore effector-target protein pairs in an unbiased manner. Here, we combined Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) for the high-throughput tagging of endogenous proteins, with generic small molecule-based protein recruitment to screen for novel proximity-based effectors. We apply this methodology in two orthogonal screens for targeted protein degradation: the first using fluorescence to monitor target protein levels directly, and the second using a cellular growth phenotype that depends on the degradation of an essential protein. Our screens revealed a multitude of potential new effector proteins for degradation and converged on members of the CTLH complex which we demonstrate potently induce degradation. Altogether, we introduce a platform for pooled induction of endogenous protein-protein interactions that can be used to expand our toolset of effector proteins for targeted protein degradation and other forms of induced proximity.

Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses.

System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a bird's-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to misfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct crosstalk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.

Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics.

The field of induced proximity therapeutics is in its ascendancy but is limited by a lack of scalable tools to systematically explore effector-target protein pairs in an unbiased manner. Here, we combined Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) for the high-throughput tagging of endogenous proteins, with generic small molecule-based protein recruitment to screen for novel proximity-based effectors. We apply this methodology in two orthogonal screens for targeted protein degradation: the first using fluorescence to monitor target protein levels directly, and the second using a cellular growth phenotype that depends on the degradation of an essential protein. Our screens revealed a multitude of potential new effector proteins for degradation and converged on members of the CTLH complex which we demonstrate potently induce degradation. Altogether, we introduce a platform for pooled induction of endogenous protein-protein interactions that can be used to expand our toolset of effector proteins for targeted protein degradation and other forms of induced proximity.

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) that cause DTNBP1, JARID2 and ASTN2 haploinsufficiencies, all of which are associated with neurological disorders. We further determine that these CNVs arose in vitro over the course of KOLF2.1J iPSC generation from a healthy donor-derived KOLF2 iPSC line and affect the expression of DNTBP1, JARID2 and ASTN2 proteins in KOLF2.1J iPSCs and neural progenitors. Therefore, our study suggests that KOLF2.1J iPSCs carry genetic variants that may be deleterious for neural cell lineages. This data is essential for a careful interpretation of neural cell studies derived from KOLF2.1J iPSCs and highlights the need for a catalogue of iPSC lines that includes a comprehensive genome characterization analysis.

Large-scale genetic screens identify BET-1 as a cytoskeleton regulator promoting actin function and life span.

The actin cytoskeleton is a three-dimensional scaffold of proteins that is a regulatory, energyconsuming network with dynamic properties to shape the structure and function of the cell. Proper actin function is required for many cellular pathways, including cell division, autophagy, chaperone function, endocytosis, and exocytosis. Deterioration of these processes manifests during aging and exposure to stress, which is in part due to the breakdown of the actin cytoskeleton. However, the regulatory mechanisms involved in preservation of cytoskeletal form and function are not well-understood. Here, we performed a multipronged, cross-organismal screen combining a whole-genome CRISPR-Cas9 screen in human fibroblasts with in vivo Caenorhabditis elegans synthetic lethality screening. We identified the bromodomain protein, BET-1, as a key regulator of actin function and longevity. Overexpression of bet-1 preserves actin function at late age and promotes life span and healthspan in C. elegans. These beneficial effects are mediated through actin preservation by the transcriptional regulator function of BET-1. Together, our discovery assigns a key role for BET-1 in cytoskeletal health, highlighting regulatory cellular networks promoting cytoskeletal homeostasis.

Impaired ribosome-associated quality control of C9orf72 arginine-rich dipeptide-repeat proteins.

Protein quality control pathways have evolved to ensure the fidelity of protein synthesis and efficiently clear potentially toxic protein species. Defects in ribosome-associated quality control and its associated factors have been implicated in the accumulation of aberrant proteins and neurodegeneration. C9orf72 repeat-associated non-AUG translation has been suggested to involve inefficient translation elongation, lead to ribosomal pausing and activation of ribosome-associated quality control pathways. However, the role of the ribosome-associated quality control complex in the processing of proteins generated through this non-canonical translation is not well understood. Here we use reporter constructs containing the C9orf72-associated hexanucleotide repeat, ribosome-associated quality control complex deficient cell models and stain for ribosome-associated quality control markers in C9orf72-expansion carrier human tissue to understand its role in dipeptide-repeat protein pathology. Our studies show that canonical ribosome-associated quality control substrates products are efficiently cleared by the ribosome-associated quality control complex in mammalian cells. Furthermore, using stalling reporter constructs, we show that repeats associated with the C9orf72-expansion induce ribosomal stalling when arginine (R)-rich dipeptide-repeat proteins are synthesized in a length-dependent manner. However, despite triggering this pathway, these arginine-rich dipeptide-repeat proteins are not efficiently processed by the core components of the ribosome-associated quality control complex (listerin, nuclear-export mediator factor and valosin containing protein) partly due to lack of lysine residues, which precludes ubiquitination. Deficient processing by this complex may be implicated in C9orf72-expansion associated disease as dipeptide-repeat protein inclusions were observed to be predominantly devoid of ubiquitin and co-localize with nuclear-export mediator factor in mutation carriers' frontal cortex and cerebellum tissue. These findings suggest that impaired processing of these arginine-rich dipeptide-repeat proteins derived from repeat-associated non-AUG translation by the ribosome-associated quality control complex may contribute to protein homeostasis dysregulation observed in C9orf72-expansion amyotrophic lateral sclerosis and frontotemporal degeneration neuropathogenesis.

Bcl-xL Enforces a Slow-Cycling State Necessary for Survival in the Nutrient-Deprived Microenvironment of Pancreatic Cancer.

Solid tumors possess heterogeneous metabolic microenvironments where oxygen and nutrient availability are plentiful (fertile regions) or scarce (arid regions). While cancer cells residing in fertile regions proliferate rapidly, most cancer cells in vivo reside in arid regions and exhibit a slow-cycling state that renders them chemoresistant. Here, we developed an in vitro system enabling systematic comparison between these populations via transcriptome analysis, metabolomic profiling, and whole-genome CRISPR screening. Metabolic deprivation led to pronounced transcriptional and metabolic reprogramming, resulting in decreased anabolic activities and distinct vulnerabilities. Reductions in anabolic, energy-consuming activities, particularly cell proliferation, were not simply byproducts of the metabolic challenge, but rather essential adaptations. Mechanistically, Bcl-xL played a central role in the adaptation to nutrient and oxygen deprivation. In this setting, Bcl-xL protected quiescent cells from the lethal effects of cell-cycle entry in the absence of adequate nutrients. Moreover, inhibition of Bcl-xL combined with traditional chemotherapy had a synergistic antitumor effect that targeted cycling cells. Bcl-xL expression was strongly associated with poor patient survival despite being confined to the slow-cycling fraction of human pancreatic cancer cells. These findings provide a rationale for combining traditional cancer therapies that target rapidly cycling cells with those that target quiescent, chemoresistant cells associated with nutrient and oxygen deprivation. SIGNIFICANCE: The majority of pancreatic cancer cells inhabit nutrient- and oxygen-poor tumor regions and require Bcl-xL for their survival, providing a compelling antitumor metabolic strategy.

Cross-species screening platforms identify EPS-8 as a critical link for mitochondrial stress and actin stabilization.

The dysfunction of mitochondria is associated with the physiological consequences of aging and many age-related diseases. Therefore, critical quality control mechanisms exist to protect mitochondrial functions, including the unfolded protein response of the mitochondria (UPRMT). However, it is still unclear how UPRMT is regulated in mammals with mechanistic discrepancies between previous studies. Here, we reasoned that a study of conserved mechanisms could provide a uniquely powerful way to reveal previously uncharacterized components of the mammalian UPRMT. We performed cross-species comparison of genetic requirements for survival under­and in response to­mitochondrial stress between karyotypically normal human stem cells and the nematode Caenorhabditis elegans. We identified a role for EPS-8/EPS8 (epidermal growth factor receptor pathway substrate 8), a signaling protein adaptor, in general mitochondrial homeostasis and UPRMT regulation through integrin-mediated remodeling of the actin cytoskeleton. This study also highlights the use of cross-species comparisons in genetic screens to interrogate cellular pathways.

Therapeutic genetic variation revealed in diverse Hsp104 homologs.

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.

Population-scale longitudinal mapping of COVID-19 symptoms, behaviour and testing.

Despite the widespread implementation of public health measures, coronavirus disease 2019 (COVID-19) continues to spread in the United States. To facilitate an agile response to the pandemic, we developed How We Feel, a web and mobile application that collects longitudinal self-reported survey responses on health, behaviour and demographics. Here, we report results from over 500,000 users in the United States from 2 April 2020 to 12 May 2020. We show that self-reported surveys can be used to build predictive models to identify likely COVID-19-positive individuals. We find evidence among our users for asymptomatic or presymptomatic presentation; show a variety of exposure, occupational and demographic risk factors for COVID-19 beyond symptoms; reveal factors for which users have been SARS-CoV-2 PCR tested; and highlight the temporal dynamics of symptoms and self-isolation behaviour. These results highlight the utility of collecting a diverse set of symptomatic, demographic, exposure and behavioural self-reported data to fight the COVID-19 pandemic.

Population-scale Longitudinal Mapping of COVID-19 Symptoms, Behavior, and Testing Identifies Contributors to Continued Disease Spread in the United States.

Despite social distancing and shelter-in-place policies, COVID-19 continues to spread in the United States. A lack of timely information about factors influencing COVID-19 spread and testing has hampered agile responses to the pandemic. We developed How We Feel, an extensible web and mobile application that aggregates self-reported survey responses, to fill gaps in the collection of COVID-19-related data. How We Feel collects longitudinal and geographically localized information on users' health, behavior, and demographics. Here we report results from over 500,000 users in the United States from April 2, 2020 to May 12, 2020. We show that self- reported surveys can be used to build predictive models of COVID-19 test results, which may aid in identification of likely COVID-19 positive individuals. We find evidence among our users for asymptomatic or presymptomatic presentation, as well as for household and community exposure, occupation, and demographics being strong risk factors for COVID-19. We further reveal factors for which users have been SARS-CoV-2 PCR tested, as well as the temporal dynamics of self- reported symptoms and self-isolation behavior in positive and negative users. These results highlight the utility of collecting a diverse set of symptomatic, demographic, and behavioral self- reported data to fight the COVID-19 pandemic.

Publisher Correction: Building an international consortium for tracking coronavirus health status.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Impaired Death Receptor Signaling in Leukemia Causes Antigen-Independent Resistance by Inducing CAR T-cell Dysfunction.

Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy.See related commentary by Green and Neelapu, p. 492.

The Hyaluronidase, TMEM2, Promotes ER Homeostasis and Longevity Independent of the UPRER.

Cells have evolved complex mechanisms to maintain protein homeostasis, such as the UPRER, which are strongly associated with several diseases and the aging process. We performed a whole-genome CRISPR-based knockout (KO) screen to identify genes important for cells to survive ER-based protein misfolding stress. We identified the cell-surface hyaluronidase (HAase), Transmembrane Protein 2 (TMEM2), as a potent modulator of ER stress resistance. The breakdown of the glycosaminoglycan, hyaluronan (HA), by TMEM2 within the extracellular matrix (ECM) altered ER stress resistance independent of canonical UPRER pathways but dependent upon the cell-surface receptor, CD44, a putative HA receptor, and the MAPK cell-signaling components, ERK and p38. Last, and most surprisingly, ectopic expression of human TMEM2 in C. elegans protected animals from ER stress and increased both longevity and pathogen resistance independent of canonical UPRER activation but dependent on the ERK ortholog mpk-1 and the p38 ortholog pmk-1.