Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes.

Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.

Spatial proteomics: a powerful discovery tool for cell biology.

Protein subcellular localization is tightly controlled and intimately linked to protein function in health and disease. Capturing the spatial proteome - that is, the localizations of proteins and their dynamics at the subcellular level - is therefore essential for a complete understanding of cell biology. Owing to substantial advances in microscopy, mass spectrometry and machine learning applications for data analysis, the field is now mature for proteome-wide investigations of spatial cellular regulation. Studies of the human proteome have begun to reveal a complex architecture, including single-cell variations, dynamic protein translocations, changing interaction networks and proteins localizing to multiple compartments. Furthermore, several studies have successfully harnessed the power of comparative spatial proteomics as a discovery tool to unravel disease mechanisms. We are at the beginning of an era in which spatial proteomics finally integrates with cell biology and medical research, thereby paving the way for unbiased systems-level insights into cellular processes. Here, we discuss current methods for spatial proteomics using imaging or mass spectrometry and specifically highlight global comparative applications. The aim of this Review is to survey the state of the field and also to encourage more cell biologists to apply spatial proteomics approaches.

SHRED Is a Regulatory Cascade that Reprograms Ubr1 Substrate Specificity for Enhanced Protein Quality Control during Stress.

When faced with proteotoxic stress, cells mount adaptive responses to eliminate aberrant proteins. Adaptive responses increase the expression of protein folding and degradation factors to enhance the cellular quality control machinery. However, it is unclear whether and how this augmented machinery acquires new activities during stress. Here, we uncover a regulatory cascade in budding yeast that consists of the hydrophilin protein Roq1/Yjl144w, the HtrA-type protease Ynm3/Nma111, and the ubiquitin ligase Ubr1. Various stresses stimulate ROQ1 transcription. The Roq1 protein is cleaved by Ynm3. Cleaved Roq1 interacts with Ubr1, transforming its substrate specificity. Altered substrate recognition by Ubr1 accelerates proteasomal degradation of misfolded as well as native proteins at the endoplasmic reticulum membrane and in the cytosol. We term this pathway stress-induced homeostatically regulated protein degradation (SHRED) and propose that it promotes physiological adaptation by reprogramming a key component of the quality control machinery.

Unbiased proteomic profiling of host cell extracellular vesicle composition and dynamics upon HIV-1 infection.

Cells release diverse types of extracellular vesicles (EVs), which transfer complex signals to surrounding cells. Specific markers to distinguish different EVs (e.g. exosomes, ectosomes, enveloped viruses like HIV) are still lacking. We have developed a proteomic profiling approach for characterizing EV subtype composition and applied it to human Jurkat T cells. We generated an interactive database to define groups of proteins with similar profiles, suggesting release in similar EVs. Biochemical validation confirmed the presence of preferred partners of commonly used exosome markers in EVs: CD81/ADAM10/ITGB1, and CD63/syntenin. We then compared EVs from control and HIV-1-infected cells. HIV infection altered EV profiles of several cellular proteins, including MOV10 and SPN, which became incorporated into HIV virions, and SERINC3, which was re-routed to non-viral EVs in a Nef-dependent manner. Furthermore, we found that SERINC3 controls the surface composition of EVs. Our workflow provides an unbiased approach for identifying candidate markers and potential regulators of EV subtypes. It can be widely applied to in vitro experimental systems for investigating physiological or pathological modifications of EV release.

Designing spiking neural networks for robust and reconfigurable computation.

Networks of spiking neurons constitute analog systems capable of effective and resilient computing. Recent work has shown that networks of symmetrically connected inhibitory neurons may implement basic computations such that they are resilient to system disruption. For instance, if the functionality of one neuron is lost (e.g., the neuron, along with its connections, is removed), the system may be robustly reconfigured by adapting only one global system parameter. How to effectively adapt network parameters to robustly perform a given computation is still unclear. Here, we present an analytical approach to derive such parameters. Specifically, we analyze k-winners-takes-all (k-WTA) computations, basic computational tasks of identifying the k largest signals from a total of N input signals from which one can construct any computation. We identify and characterize different dynamical regimes and provide analytical expressions for the transitions between different numbers k of winners as a function of both input and network parameters. Our results thereby provide analytical insights about the dynamics underlying k-winner-takes-all functionality as well as an effective way of designing spiking neural network computing systems implementing disruption-resilient dynamics.

Revealing system dimension from single-variable time series.

Complex and networked dynamical systems characterize the time evolution of most of the natural and human-made world. The dimension of their state space, i.e., the number of (active) variables in such systems, arguably constitutes their most fundamental property yet is hard to access in general. Recent work [Haehne et al., Phys. Rev. Lett. 122, 158301 (2019)] introduced a method of inferring the state space dimension of a multi-dimensional networked system from repeatedly measuring time series of only some fraction of observed variables, while all other variables are hidden. Here, we show how time series observations of one single variable are mathematically sufficient for dimension inference. We reveal how successful inference in practice depends on numerical constraints of data evaluation and on experimental choices, in particular the sampling intervals and the total duration of observations. We illustrate robust inference for systems of up to N=10 to N=100 variables by evaluating time series observations of a single variable. We discuss how the faithfulness of the inference depends on the quality and quantity of collected data and formulate some general rules of thumb on how to approach the measurement of a given system.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

Organellar Maps Through Proteomic Profiling - A Conceptual Guide.

Protein subcellular localization is an essential and highly regulated determinant of protein function. Major advances in mass spectrometry and imaging have allowed the development of powerful spatial proteomics approaches for determining protein localization at the whole cell scale. Here, a brief overview of current methods is presented, followed by a detailed discussion of organellar mapping through proteomic profiling. This relatively simple yet flexible approach is rapidly gaining popularity, because of its ability to capture the localizations of thousands of proteins in a single experiment. It can be used to generate high-resolution cell maps, and as a tool for monitoring protein localization dynamics. This review highlights the strengths and limitations of the approach and provides guidance to designing and interpreting profiling experiments.

Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval.

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

Molecular Basis for the Interaction Between AP4 ß4 and its Accessory Protein, Tepsin.

The adaptor protein 4 (AP4) complex (ϵ/ß4/µ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 ß4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal ß4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the ß4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on ß4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function.

A novel disorder reveals clathrin heavy chain-22 is essential for human pain and touch development.

Congenital inability to feel pain is very rare but the identification of causative genes has yielded significant insights into pain pathways and also novel targets for pain treatment. We report a novel recessive disorder characterized by congenital insensitivity to pain, inability to feel touch, and cognitive delay. Affected individuals harboured a homozygous missense mutation in CLTCL1 encoding the CHC22 clathrin heavy chain, p.E330K, which we demonstrate to have a functional effect on the protein. We found that CLTCL1 is significantly upregulated in the developing human brain, displaying an expression pattern suggestive of an early neurodevelopmental role. Guided by the disease phenotype, we investigated the role of CHC22 in two human neural crest differentiation systems; human induced pluripotent stem cell-derived nociceptors and TRKB-dependant SH-SY5Y cells. In both there was a significant downregulation of CHC22 upon the onset of neural differentiation. Furthermore, knockdown of CHC22 induced neurite outgrowth in neural precursor cells, which was rescued by stable overexpression of small interfering RNA-resistant CHC22, but not by mutant CHC22. Similarly, overexpression of wild-type, but not mutant, CHC22 blocked neurite outgrowth in cells treated with retinoic acid. These results reveal an essential and non-redundant role for CHC22 in neural crest development and in the genesis of pain and touch sensing neurons.

Adaptor protein complexes AP-4 and AP-5: new players in endosomal trafficking and progressive spastic paraplegia.

The adaptor proteins (APs) are a family of five heterotetrameric complexes with important functions in vesicle trafficking. While the roles of APs 1-3 are broadly established, comparatively little is known about AP-4 and AP-5. Current evidence suggests that AP-4 mediates TGN to endosome transport of specific cargo proteins, such as the amyloid precursor protein APP, and that it is involved in basolateral sorting in polarized cells. Furthermore, several independent studies have reported human patients with mutations in AP-4 genes. AP-4 deficiency causes severe intellectual disability and progressive spastic para- or tetraplegia, supporting an important role for AP-4 in brain function and development. The newly discovered AP-5 complex appears to be involved in endosomal dynamics; its precise localization and function are still unclear. Intriguingly, AP-5 deficiency is also associated with progressive spastic paraplegia, suggesting that AP-5, like AP-4, plays a fundamental role in neuronal development and homeostasis. The unexpected phenotypic parallels between AP-4 and AP-5 patients may in turn suggest a functional relationship of the two APs in vesicle trafficking.

High-content screening identifies a small molecule that restores AP-4-dependent protein trafficking in neuronal models of AP-4-associated hereditary spastic paraplegia.

Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adapter protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, BCH-HSP-C01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate potential mechanisms of action of BCH-HSP-C01. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future studies.

Deep and fast label-free Dynamic Organellar Mapping.

The Dynamic Organellar Maps (DOMs) approach combines cell fractionation and shotgun-proteomics for global profiling analysis of protein subcellular localization. Here, we enhance the performance of DOMs through data-independent acquisition (DIA) mass spectrometry. DIA-DOMs achieve twice the depth of our previous workflow in the same mass spectrometry runtime, and substantially improve profiling precision and reproducibility. We leverage this gain to establish flexible map formats scaling from high-throughput analyses to extra-deep coverage. Furthermore, we introduce DOM-ABC, a powerful and user-friendly open-source software tool for analyzing profiling data. We apply DIA-DOMs to capture subcellular localization changes in response to starvation and disruption of lysosomal pH in HeLa cells, which identifies a subset of Golgi proteins that cycle through endosomes. An imaging time-course reveals different cycling patterns and confirms the quantitative predictive power of our translocation analysis. DIA-DOMs offer a superior workflow for label-free spatial proteomics as a systematic phenotype discovery tool.

Perforin-2 is a pore-forming effector of endocytic escape in cross-presenting dendritic cells.

During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1-/- mice failed to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.

High-Content Small Molecule Screen Identifies a Novel Compound That Restores AP-4-Dependent Protein Trafficking in Neuronal Models of AP-4-Associated Hereditary Spastic Paraplegia.

Unbiased phenotypic screens in patient-relevant disease models offer the potential to detect novel therapeutic targets for rare diseases. In this study, we developed a high-throughput screening assay to identify molecules that correct aberrant protein trafficking in adaptor protein complex 4 (AP-4) deficiency, a rare but prototypical form of childhood-onset hereditary spastic paraplegia, characterized by mislocalization of the autophagy protein ATG9A. Using high-content microscopy and an automated image analysis pipeline, we screened a diversity library of 28,864 small molecules and identified a lead compound, C-01, that restored ATG9A pathology in multiple disease models, including patient-derived fibroblasts and induced pluripotent stem cell-derived neurons. We used multiparametric orthogonal strategies and integrated transcriptomic and proteomic approaches to delineate putative molecular targets of C-01 and potential mechanisms of action. Our results define molecular regulators of intracellular ATG9A trafficking and characterize a lead compound for the treatment of AP-4 deficiency, providing important proof-of-concept data for future Investigational New Drug (IND)-enabling studies.

AP-4-mediated axonal transport controls endocannabinoid production in neurons.

The adaptor protein complex AP-4 mediates anterograde axonal transport and is essential for axon health. AP-4-deficient patients suffer from a severe neurodevelopmental and neurodegenerative disorder. Here we identify DAGLB (diacylglycerol lipase-beta), a key enzyme for generation of the endocannabinoid 2-AG (2-arachidonoylglycerol), as a cargo of AP-4 vesicles. During normal development, DAGLB is targeted to the axon, where 2-AG signalling drives axonal growth. We show that DAGLB accumulates at the trans-Golgi network of AP-4-deficient cells, that axonal DAGLB levels are reduced in neurons from a patient with AP-4 deficiency, and that 2-AG levels are reduced in the brains of AP-4 knockout mice. Importantly, we demonstrate that neurite growth defects of AP-4-deficient neurons are rescued by inhibition of MGLL (monoacylglycerol lipase), the enzyme responsible for 2-AG hydrolysis. Our study supports a new model for AP-4 deficiency syndrome in which axon growth defects arise through spatial dysregulation of endocannabinoid signalling.

Spatial centrosome proteome of human neural cells uncovers disease-relevant heterogeneity.

The centrosome provides an intracellular anchor for the cytoskeleton, regulating cell division, cell migration, and cilia formation. We used spatial proteomics to elucidate protein interaction networks at the centrosome of human induced pluripotent stem cell-derived neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell type-specific, with protein hubs involved in RNA dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant pre-mRNA-processing factor 6 (PRPF6) reproduced the periventricular misplacement in the developing mouse brain, highlighting missplicing of transcripts of a microtubule-associated kinase with centrosomal location as essential for the phenotype. Collectively, cell type-specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes.

Comparative proteomics of clathrin-coated vesicles.

Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a "mock CCV" fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.