Biology subjects

Ward, M. E.

Publications and source records attributed to Ward, M. E..

31 records · Page 2Linked to original sources

bioRxiv2021

A fully automated FAIMS-DIA proteomic pipeline for high-throughput characterization of iPSC-derived neurons

Fully automated proteomic pipelines have the potential to achieve deep coverage of cellular proteomes with high throughput and scalability. However, it is important to evaluate performance, including both reproducibility and ability to provide meaningful levels of biological insight. Here, we present an approach combining high field asymmetric waveform ion mobility spectrometer (FAIMS) interface and data independent acquisition (DIA) proteomics approach developed as part of the induced pluripotent stem cell (iPSC) Neurodegenerative Disease Initiative (iNDI), a large-scale effort to understand how inherited diseases may manifest in neuronal cells. Our FAIMS-DIA approach identified more than 8000 proteins per mass spectrometry (MS) acquisition as well as superior total identification, reproducibility, and accuracy compared to other existing DIA methods. Next, we applied this approach to perform a longitudinal proteomic profiling of the differentiation of iPSC-derived neurons from the KOLF2.1J parental line used in iNDI. This analysis demonstrated a steady increase in expression of mature cortical neuron markers over the course of neuron differentiation. We validated the performance of our proteomics pipeline by comparing it to single cell RNA-Seq datasets obtained in parallel, confirming expression of key markers and cell type annotations. An interactive webapp of this temporal data is available for aligned-UMAP visualization and data browsing (https://share.streamlit.io/anant-droid/singlecellumap). In summary, we report an extensively optimized and validated proteomic pipeline that will be suitable for large-scale studies such as iNDI.

neuroscience↗

bioRxiv2021

Deficiency of the lysosomal protein CLN5 alters lysosomal function and movement

Batten disease is a devastating childhood rare neurodegenerative disease characterized by rapid deterioration of cognition and movement, leading to death within ten to thirty years of age. One of the thirteen Batten disease forms, CLN5 Batten disease, is caused by mutations in the CLN5 gene leading to motor deficits, mental deterioration, cognitive impairment, visual impairment, and epileptic seizures in children. A characteristic pathology in CLN5 Batten disease is the defects in lysosomes, leading to neuronal dysfunction. In this study, we aimed to investigate the lysosomal changes in CLN5-deficient human neurons. We used an induced pluripotent stem cell system, which generates pure human cortical-like glutamatergic neurons. Using CRISPRi, we inhibited the expression of CLN5 in human neurons. The CLN5-deficient human neurons showed neutralised lysosomal acidity and reduced lysosomal enzyme activity measured by microscopy and flow cytometry. Furthermore, the CLN5-deficient human neurons also showed impaired lysosomal movement - a phenotype that has never been reported in CLN5 Batten disease. Lysosomal trafficking is key to maintain local degradation of cellular wastes, especially in long neuronal projections and our results from the human neuronal model present a key finding to understand the underlying lysosomal pathology in neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Directed Differentiation of Human Pluripotent Stem Cells into Radial Glia and Astrocytes Bypasses Neurogenesis

Derivation of astrocytes from human pluripotent stem cells (hPSCs) is inefficient and cumbersome, impeding their use in biomedical research. Here, we developed a highly efficient chemically defined astrocyte differentiation strategy that overcomes current limitations. This approach largely bypasses neurogenesis, which otherwise precedes astrogliogenesis during brain development and in vitro experiments. hPSCs were first differentiated into radial glial cells (RGCs) exhibiting in vivo-like radial glia signatures. Activation of NOTCH and JAK/STAT pathways in bona fide RGCs resulted in direct astrogliogenesis confirmed by expression of various glial markers (NFIA, NFIB, SOX9, CD44, S100B, GFAP). Transcriptomic and genome-wide epigenetic analyses confirmed RGC-to-astrocyte differentiation and absence of neurogenesis. The morphological and functional identity of hPSC-derived astrocytes was confirmed by using an array of methods (e.g. electron microscopy, calcium imaging, co-culture with neurons, grafting into mouse brains). Lastly, the scalable protocol was adapted to a robotic platform and used to model Alexander disease. In conclusion, our findings uncover remarkable plasticity in neural lineage progression that can be exploited to manufacture large numbers of human hPSC-derived astrocytes for drug development and regenerative medicine.

developmental biology↗

bioRxiv2021

Myotubularin-related phosphatase 5 is a critical determinant of autophagy in neurons

Autophagy is a conserved, multi-step process of capturing proteolytic cargo in autophagosomes for lysosome degradation. The capacity to remove toxic proteins that accumulate in neurodegenerative disorders attests to the disease-modifying potential of the autophagy pathway. However, neurons respond only marginally to conventional methods for inducing autophagy, limiting efforts to develop therapeutic autophagy modulators for neurodegenerative diseases. The determinants underlying poor autophagy induction in neurons and the degree to which neurons and other cell types are differentially sensitive to autophagy stimuli are incompletely defined. Accordingly, we sampled nascent transcript synthesis and stabilities in fibroblasts, induced pluripotent stem cells (iPSCs) and iPSC-derived neurons (iNeurons), thereby uncovering a neuron-specific stability of transcripts encoding myotubularin-related phosphatase 5 (MTMR5). MTMR5 is an autophagy suppressor that acts with its binding partner, MTMR2, to dephosphorylate phosphoinositides critical for autophagy initiation and autophagosome maturation. We found that MTMR5 is necessary and sufficient to suppress autophagy in iNeurons and undifferentiated iPSCs. Using optical pulse labeling to visualize the turnover of endogenously-encoded proteins in live cells, we observed that knockdown of MTMR5 or MTMR2, but not MTMR9, significantly enhances neuronal degradation of TDP-43, an autophagy substrate implicated in several neurodegenerative diseases. Accordingly, our findings establish a regulatory mechanism of autophagy intrinsic to neurons and targetable for clearing disease-related proteins in a cell type-specific manner. In so doing, our results not only unravel novel aspects of neuronal biology and proteostasis, but also elucidate a strategy for modulating neuronal autophagy that could be of high therapeutic potential for multiple neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Tau interactome mapping reveals dynamic processes in synapses and mitochondria associated with neurodegenerative disease

Tau (MAPT) drives neuronal dysfunction in Alzheimers disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established activity-dependent interactions of Tau with presynaptic vesicle proteins during Tau secretion and mapped the exact APEX-tau-induced biotinylated tyrosines to the cytosolic domains of the interacting vesicular proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Taus interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multi-modal and dynamic Tau interactomes with unprecedented spatiotemporal resolution shed novel insights into Taus role in neuronal function and disease-related processes with potential therapeutic targets to block Tau-mediated pathogenesis.

neuroscience↗

bioRxiv2021

A CRISPRi/a platform in iPSC-derived microglia uncovers regulators of disease states

Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human iPSC-derived microglia. We developed an efficient eight-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the "druggable genome". These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by SPP1 expression was selectively depleted by CSF1R inhibition. Thus, our platform can systematically uncover regulators of microglia states, enabling their functional characterization and therapeutic targeting.

neuroscience↗

bioRxiv2021

Common ALS/FTD risk variants in UNC13A exacerbate its cryptic splicing and loss upon TDP-43 mislocalization

Variants within the UNC13A gene have long been known to increase risk of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-43. Here, we show that TDP-43 depletion induces robust inclusion of a cryptic exon (CE) within UNC13A, a critical synaptic gene, resulting in nonsense-mediated decay and protein loss. Strikingly, two common polymorphisms strongly associated with ALS/FTD risk directly alter TDP-43 binding within the CE or downstream intron, increasing CE inclusion in cultured cells and in patient brains. Our findings, which are the first to demonstrate a genetic link specifically between loss of TDP-43 nuclear function and disease, reveal both the mechanism by which UNC13A variants exacerbate the effects of decreased nuclear TDP-43 function, and provide a promising therapeutic target for TDP-43 proteinopathies. One-Sentence SummaryShared ALS/FTD risk variants increase the sensitivity of a cryptic exon in the synaptic gene UNC13A to TDP-43 depletion.

neuroscience↗

bioRxiv2020

Neuronal Enhancers are Hotspots For DNA Single-Strand Break Repair

Genome stability is essential for all cell types. However, defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons. The neuronal genome is subjected to a constant barrage of endogenous DNA damage due to high levels of oxidative metabolism in the central nervous system. Surprisingly, we know little about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here, we show that neurons, but not other post-mitotic cells, accumulate unexpectedly high numbers of DNA single-strand breaks (SSBs) at specific sites within the genome. These recurrent SSBs are found within enhancers, and trigger DNA repair through recruitment and activation of poly(ADP-ribose) polymerase-1 (PARP1) and XRCC1, the central SSB repair scaffold protein. Notably, deficiencies in PARP1, XRCC1, or DNA polymerase {beta} elevate the localized incorporation of nucleotides, suggesting that the ongoing DNA synthesis at neuronal enhancers involves both short-patch and long-patch SSB repair processes. These data reveal unexpected levels of localized and continuous DNA single-strand breakage in neurons, suggesting an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.

neuroscience↗

bioRxiv2020

Development and Comparative Evaluation of Endolysosomal Proximity Labeling-based Proteomic Methods in Human iPSC-derived Neurons

Proximity-based in situ labeling techniques offer a unique way to capture both stable and transient protein-protein and protein-organelle interactions. Combining this technology with mass spectrometry (MS)-based proteomics allows us to obtain snapshots of molecular microenvironments with nanometer resolution, facilitating the discovery of complex and dynamic protein networks. However, a number of technical challenges still exist, such as interferences from endogenously biotinylated proteins and other highly abundant bystanders, how to select the proper controls to minimize false discoveries, and experimental variations among biological/technical replicates. Here, we developed a new method to capture the proteomic microenvironment of the neuronal endolysosomal network, by knocking in (KI) an engineered ascorbate peroxidase (APEX) gene to the endogenous locus of lysosome-associated membrane protein 1 (LAMP1). We found that normalizing proximity labeling proteomics data to the endogenously biotinylated protein (PCCA) can greatly reduce variations and enable fair comparisons among different batch of APEX labeling and different APEX probes. We conducted comparative evaluation between this KI-LAMP1-APEX method and our two overexpression LAMP1-APEX probes, achieving complementary coverage of both known and new lysosomal membrane and lysosomal-interacting proteins in human iPSC-derived neurons. To summarize, this study demonstrated new analytical tools to characterize lysosomal functions and microenvironment in human neurons and filled critical gaps in the field for designing and optimizing proximity labeling proteomic experiments.

systems biology↗

bioRxiv2020

Image-based pooled whole genome CRISPR screening for Parkin and TFEB subcellular localization

Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9 (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated, isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty of the sixty-four hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-Photoswitchable Screening (AI-PS) offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2019

Selective autophagic clearance of protein aggregates is mediated by the autophagy receptor, TAX1BP1

Misfolded protein aggregates can disrupt cellular homeostasis and cause toxicity, a hallmark of numerous neurodegenerative diseases. Protein quality control by the ubiquitin proteasome system (UPS) and autophagy is vital for clearance of aggregates and maintenance of cellular homeostasis1. Autophagy receptor proteins bridge the interaction between ubiquitinated proteins and the autophagy machinery allowing selective elimination of cargo2. Aggrephagy is critical to protein quality control, but how aggregates are recognized and targeted for degradation is not well understood. Here we examine the requirements for 5 autophagy receptor proteins: OPTN, NBR1, p62, NDP52, and TAX1BP1 in proteotoxic stress-induced aggregate clearance. Endogenous TAX1BP1 is both recruited to and required for the clearance of stress-induced aggregates while overexpression of TAX1BP1 increases aggregate clearance through autophagy. Furthermore, TAX1BP1 depletion sensitizes cells to proteotoxic stress and Huntingtons disease-linked polyQ proteins, whereas TAX1BP1 overexpression clears cells of polyQ protein aggregates by autophagy. We propose a broad role for TAX1BP1 in the clearance of cytotoxic proteins, thus identifying a new mode of clearance of protein inclusions.

cell biology↗

bioRxiv2019

CRISPR-based platform for multimodal genetic screens in human iPSC-derived neurons

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons, and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons.

cell biology↗

bioRxiv2018

Conformational dynamics of a photosynthetic light-harvesting complex in native thylakoid membranes

Photosynthetic light-harvesting complexes of higher plants, moss and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr Light-Harvesting Complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states. STATEMENT OF SIGNIFICANCEPhotosynthetic Light-Harvesting Complexes undergo dynamic conformational transitions that regulate the capacity of the light-harvesting antenna. We demonstrate the application of solid-state (ss)NMR spectroscopy to investigate the structural dynamics of LHCII, the most abundant LHC complex of plants and algae, in native membranes. Selective dynamic protein and pigment residues are identified that are putative sites for a conformational switch.

biophysics↗