Biology subjects

Zhou, R.

Publications and source records attributed to Zhou, R..

At least 91 records · Page 5Linked to original sources

bioRxiv2024

Deciphering gene regulatory programs in mouse embryonic skin through single-cell multiomics analysis

BackgroundCell type-specific transcriptional heterogeneity in embryonic mouse skin is well-documented, but few studies have investigated the regulatory mechanisms. ResultsHere, we present high throughput single-cell chromatin accessibility and transcriptome sequencing (HT-scCAT-seq), a method that simultaneously profiles transcriptome and chromatin accessibility. We utilized HT-scCAT-seq to dissect the gene regulatory mechanism governing epidermal stratification, periderm terminal differentiation, and fibroblast specification. ConclusionsBy linking chromatin accessibility to gene expression, we identified candidate cis-regulatory elements (cCREs) and target genes crucial for dermal and epidermal development. We described cells with similar gene expression profiles that exhibit distinct chromatin accessibility statuses during periderm terminal differentiation. Finally, we characterized the underlying lineage-determining transcription factors (TFs), and demonstrated that ALX4 and RUNX2 were candidate TF regulators of the dermal papilla lineage development through in silico perturbation analysis.

developmental biology↗

bioRxiv2024

Pipeline for Assessing Tumor Immune Status Using Superplex Immunostaining and Spatial Immune Interaction Analysis

The characteristics of the tumor microenvironment (TME) are closely linked to tumor progression and treatment response. The TME comprises various cell types, their spatial distribution, cell-cell interactions, and their organization into cellular niches or neighborhoods. To capture this complexity, several spatial profiling technologies have been developed. However, challenges such as low throughput, high costs, and complicated data analysis have limited their widespread use in immune research. In this study, we introduce the Cyclic-multiplex TSA (CmTSA) staining platform, a high-throughput superplex staining technology based on tyramide signal amplification (TSA) immunostaining combined with an efficient fluorophore recycling method. The CmTSA platform allows for the labeling of 30-60 antigens across multiple parallel formalin-fixed paraffin-embedded (FFPE) slides. Furthermore, the automated CmTSA workflow requires only standard histological equipment and conventional immunohistochemistry (IHC) primary antibodies (Abs), significantly reducing costs. While the superplex images produced contain extensive multidimensional information, extracting the spatial features of the TME from raw pixel data can be challenging. To address this, we present a computer vision-based analysis pipeline, which begins with deep learning-based algorithms to segment individual cells and identify cell types based on defined annotation rules. It then evaluates the spatial distribution tendencies of each cell type, the interaction intensity between paired cells, and the multicellular functional niches. This comprehensive approach enables researchers to visualize and quantify the types, states, and levels of immune activities within the TME effectively, advancing tumor immunology research and precision immune medicine.

cancer biology↗

bioRxiv2024

Multi-strategies embedded framework for neoantigen vaccine maturation

Effective cancer immunotherapy hinges on the precise recognition of neoantigens, presented as binary complexes with major histocompatibility complex (MHC) molecules, by T cell receptors (TCR). The development of immunogenic peptide predictors and generators plays a central role in personalizing immunotherapies while reducing experimental costs. However, the current methods often fall short in leveraging structural data efficiently and providing comprehensive guidance for neoantigen selection. To address these limitations, we introduce NEOM, a novel neoantigen maturation framework encompassing five distinct modules: "policy", "structure", "evaluation", "selection" and "filter". This framework is designed to enhance precision, interpretability, customizability and cost-effectiveness in neoantigen screening. We evaluated NEOM using a set of random synthetic peptides, followed by available clinically-derived peptides. NEOM achieved higher performance on generated peptide quality compared to other baseline models. Using established predictors for filtering revealed a substantial number of peptides with immunogenic potential. Subsequently, a more rigorous binding affinity evaluation using free energy perturbation methods identified 6 out of 38 candidates showing superior binding characteristics. MHC tetramer peptide exchange assays and flow cytometry experiments further validate five of them. These results demonstrate that NEOM not only excels in identifying diverse peptides with enhanced binding stability and affinity for MHC molecules but also augments their immunogenic potential, showcasing its utility in advancing personalized immunotherapies.

bioinformatics↗

bioRxiv2024

PerturbAtlas: A Comprehensive Atlas of Public Genetic Perturbation Bulk RNA-seq Datasets

Manipulating gene expression is crucial for understanding gene function, with high-throughput sequencing techniques such as RNA-seq elucidating the downstream mechanisms involved. However, the lack of a standardized metadata format for small-scale perturbation expression datasets in public repositories hinders their reuse. To address this issue, we developed PerturbAtlas, an add-value resource that re-analyzes publicly archived RNA-seq libraries to provide quantitative data on gene expression, transcript profiles, and alternative splicing events following genetic perturbation. PerturbAtlas assists users in identifying trends at the gene and isoform levels in perturbation assays by re-analyzing a curated set of 122,801 RNA-seq libraries across 13 species. This resource is freely available at https://perturbatlas.kratoss.site as both raw data tables and an interactive browser, allowing searches by species, tissue, or genomic features. The results provide detailed information on alterations following perturbations, accessible through both forward and reverse approaches, thereby enabling the exploration of perturbation consequences and the identification of potential causal perturbations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/605482v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@150012org.highwire.dtl.DTLVardef@b7ac25org.highwire.dtl.DTLVardef@86c4feorg.highwire.dtl.DTLVardef@1ef3341_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

IsRNAcirc: 3D structure prediction of circular RNAs based on coarse-grained molecular dynamics simulation

As an emerging class of RNA molecules, circular RNAs play pivotal roles in various biological processes, thereby determining their three-dimensional (3D) structure is crucial for a deep understanding of their biological significances. Similar to linear RNAs, the development of computational methods for circular RNA 3D structure prediction is challenging, especially considering the inherent flexibility and potentially long length of circular RNAs. Here, we introduce an extension of our previous IsRNA2 model, named IsRNAcirc, to enable circular RNA 3D structure predictions through coarse-grained molecular dynamics simulations. The workflow of IsRNAcirc consists of four main steps, including input preparation, end closure, structure prediction, and model refinement. Our results demonstrate that IsRNAcirc can provide reasonable 3D structure predictions for circular RNAs, which significantly reduce the locally irrational elements contained in the initial input. Moreover, for a validation test set comprising 34 circular RNAs, our IsRNAcirc can generate 3D models with better scores than the template-based 3dRNA method. These findings demonstrate that our IsRNAcirc method is a promising tool to explore the structural details along with intricate interactions of circular RNAs. Author SummaryKnowledge of the 3D structure of circular RNA molecules can help us better understand their roles in eukaryotic cells. However, experimental determination of circular RNA 3D structure remains challenging, with currently only one complete circular RNA 3D structure available in the Protein Data Bank. Thus, extending from their linear counterparts, computational methods that can predict circular RNA 3D structure are urgently needed. In particular, physics-based de novo 3D structure prediction methods are promising due to their powerful sampling capability in the conformational space, which avoid the problem of limited available templates in template-based approaches. We have developed IsRNAcirc, a new physics-based method that employs an accurate coarse-grained force field and replica-exchange molecular dynamics simulations to predict the 3D structure of circular RNAs. IsRNAcirc is shown to significantly reduce the number of locally irrational structures in the input trials, which are caused by the lack of suitable templates and the improper assembly of fragments. Furthermore, in the absence of experimental reference structures, the predicted 3D models were validated using the popular scoring functions, such as DFIRE-RNA and rsRNASP, demonstrating that IsRNAcirc outperforms the template-based 3dRNA method in generating low-energy conformations.

biophysics↗

bioRxiv2024

Reversible control of kinase signaling through chemical-induced dephosphorylation

The coordination between kinases and phosphatases is crucial for regulating the phosphorylation levels of essential signaling molecules. Methods enabling precise control of kinase activities are valuable for understanding the kinase functions and for developing targeted therapies. Here, we use the abscisic acid (ABA)-induced proximity system to reversibly control kinase signaling by recruiting phosphatases. Using this method, we found that the oncogenic tyrosine kinase BCR::ABL1 can be inhibited by recruiting various cytoplasmic phosphatases. We also discovered that the oncogenic serine/threonine kinase BRAF(V600E), which has been reported to bypass phosphorylation regulation, can be positively regulated by protein phosphatase 1 (PP1) and negatively regulated by PP5. Additionally, we observed that the dual-specificity kinase MEK1 can be inhibited by recruiting PP5. This suggests that bifunctional molecules capable of recruiting PP5 to MEK or RAF kinases could be promising anticancer drug candidates. Thus, the ABA-induced dephosphorylation method enables rapid screening of phosphatases to precisely control kinase signaling.

cancer biology↗

bioRxiv2024

25-hydroxycholesterol dysregulates brain endothelial cell function and exacerbates cerebral haemorrhage

The antiviral enzyme cholesterol 25-hydroxylase (CH25H) and its metabolite 25-hydroxycholesterol (25HC), which modulates cholesterol metabolism during infection, have been previously associated with vascular pathology. Viral infections have been linked to risk of intracerebral haemorrhage (ICH) but the molecular mechanisms leading to brain vessel rupture via antiviral responses remain unknown. We hypothesised that the CH25H/25HC pathway may impact neuroendothelial integrity in the context of infection-associated ICH. Here, using a SARS-CoV-2-spike-induced zebrafish ICH model and foetal human SARS-CoV-2-associated cortical tissue containing microbleeds, we identified an upregulation of CH25H in infection-associated cerebral haemorrhage. Using zebrafish ICH models and human brain endothelial cells, we asked whether 25HC may promote neurovascular dysfunction by modulating cholesterol metabolism. We found that 25HC and pharmacological inhibition of HMGCR by atorvastatin interacted to exacerbate brain bleeding in zebrafish larvae and in vitro brain endothelial dysfunction. In vitro 25HC-induced dysfunction was also rescued by cholesterol supplementation. These results demonstrate that the antiviral factor 25HC can dysregulate brain endothelial function by remodelling cholesterol metabolism. We propose that the CH25H/25HC pathway represents an important component in the pathophysiology of brain vessel dysfunction associated with infection and cholesterol dysregulation in the context of ICH. Summary StatementThe antiviral metabolite 25-hydroxycholesterol dysregulates brain endothelial function by remodelling cholesterol metabolism, thereby providing a mechanistic link between viral infection and brain endothelial dysfunction in conditions such as intracerebral haemorrhage.

neuroscience↗

bioRxiv2024

Plasticity and Dynamics of Hematopoietic Cells within Bone Marrow Microenvironment in Leukemia

Extensive research has been conducted on the plasticity of malignant cells and nonmalignant cells in solid tumor. However, the plasticity of bone marrow hematopoietic cells in leukemia have remained largely unexplored. In this study, we aimed to investigate cell changings in hematopoietic cells through lineage tracing across various types of leukemias. We had compiled a landscape of leukemia and constructed phylogenetic trees of hematopoietic cells through utilizing massively parallel scRNA-seq data, mtDNA mutation and SNP analysis. Based on the observed cell changings, we identified several types of cell changings, including transdifferentiation, dedifferentiation, and state transition, except for differentiation and expansion. In AML and CMML, GMPs and neutrophils showed a higher potential for transferring to other cell types. In BPDCN, pDCs were less prone to switching to other cell types, while T cells demonstrated high plasticity. In B-ALL and B-CLL, B-ALL blast cells and B-CLL blast cells emerged at the most dynamic state. The dynamics of hematopoietic cells in AML, BPDCN and ALL changed along with the clinical process. Extrinsic factors within the leukemia microenvironment may influence the cell changings. Regulons encountered an intermediate cell state during the process of transition to myeloid cells and erythroid cells. We also found a correlation between B-common blast cells and T cells, suggesting a potential transition from B lymphoblastic leukemia to T lymphoblastic leukemia. In conclusion, our study unveiled the distinct plasticity and dynamics of hematopoietic cells in various types of leukemia. This sheds light on the possibility of targeting cell changes as a new strategy for leukemia treatment and improving current immunotherapy.

bioinformatics↗

bioRxiv2024

Cell-specific polymerization-driven biomolecular condensate formation fine-tunes root tissue morphogenesis

Formation of biomolecular condensates can be driven by weak multivalent interactions and emergent polymerization. However, the mechanism of polymerization-mediated condensate formation is less studied. We found lateral root cap cell (LRC)-specific SUPPRESSOR OF RPS4-RLD1 (SRFR1) condensates fine-tune primary root development. Polymerization of the SRFR1 N-terminal domain is required for both LRC condensate formation and optimal root growth. Surprisingly, the first intrinsically disordered region (IDR1) of SRFR1 can be functionally substituted by a specific group of intrinsically disordered proteins known as dehydrins. This finding facilitated the identification of functional segments in the IDR1 of SRFR1, a generalizable strategy to decode unknown IDRs. With this functional information we further improved root growth by modifying the SRFR1 condensation module, providing a strategy to improve plant growth and resilience.

molecular biology↗

bioRxiv2024

SCAPE-APA: a package for estimating alternative polyadenylation events from scRNA-seq data

SummarySCAPE is a package we previously developed to estimate alternative polyadenylation events from single cell RNA-seq (scRNA-seq) data, which is composed of ad-hoc python scripts and has speed issues when handling large scRNA-seq data. To suit the needs of analyzing large scRNA-seq datasets, we present SCAPE-APA, which is a re-implementation of SCAPE with substantial changes. We made the following updates to the package (1) we binned similar reads together to accelerate the estimation (2) we re-derived the mixture model to tailor it for binned reads (3) we implemented the inference algorithm using Taichi language for acceleration (4) we re-implemented the untranslated region (UTR) annotation extraction script using the professional package gffutils for better maintenance (5) we wrote a script to detect spurious alternative polyadenylation sites generated due to junction reads and (6) we made a formal python package and uploaded it to the Python Package Index website (Pypi). Availability and ImplementationScape-apa is freely available at https://github.com/chengl7-lab/scape and can be easily installed using pip. Contactlu.cheng.ac@gmail.com

bioinformatics↗

bioRxiv2023

Disruption of redox balance in glutaminolytic triple negative breast cancer by inhibition of glutamate export and glutaminase

In triple-negative breast cancer (TNBC) that relies on catabolism of amino acid glutamine, glutaminase (GLS) converts glutamine to glutamate, which facilitates glutathione synthesis by mediating the enrichment of intracellular cystine via xCT antiporter activity. To overcome chemo resistant TNBC, we have tested a strategy of disrupting cellular redox balance by inhibition of GLS and xCT by CB839 and Erastin, respectively. Key findings of our study include: 1. Dual metabolic inhibition (CB839+Erastin) led to significant increases of cellular superoxide level in both parent and chemo resistant TNBC cells, but superoxide level was distinctly lower in resistant cells. 2. Dual metabolic inhibition combined with doxorubicin or cisplatin induced significant apoptosis in TNBC cells and is associated with high degrees of GSH depletion. In vivo, dual metabolic inhibition plus cisplatin led to significant growth delay of chemo resistant human TNBC xenografts. 3. Ferroptosis is induced by doxorubicin (DOX) but not by cisplatin or paclitaxel. Addition of dual metabolic inhibition to DOX chemotherapy significantly enhanced ferroptotic cell death. 4. Significant changes in cellular metabolites concentration preceded transcriptome changes revealed by single cell RNA sequencing, underscoring the potential of capturing early changes in metabolites as pharmacodynamic markers of metabolic inhibitors. Here we demonstrated that 4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG) PET detected xCT blockade by Erastin or its analog in mice bearing human TNBC xenografts. In summary, our study provides compelling evidence for the therapeutic benefit and feasibility of non-invasive monitoring of dual metabolic blockade as a translational strategy to sensitize chemo resistant TNBC to cytotoxic chemotherapy.

cancer biology↗

bioRxiv2023

Accurate 3D SMLM localization via Vectorial In-situ PSF Retrieval and Aberration Assessment

In single-molecule localization microscopy (SMLM), achieving precise localization hinges on obtaining an authentic point spread function (PSF) influenced by system and sample-induced aberrations. Here, we introduce VISPR (Vectorial in-situ PSF retrieval) retrieving precise 3D PSF models considering both system and sample-induced aberrations under SMLM conditions. By employing the theory of vectorial PSF model and maximum likelihood estimation (MLE) phase retrieval, VISPR is capable of reconstructing an accurate 3D PSF model achieving the theoretically minimum uncertainty and accurately reflecting three-dimensional information of single molecules. This capability empowers accurate 3D super-resolution reconstruction in 3D SMLM. Additionally, VISPR applies to low signal-to-noise ratio circumstances and is adept at retrieving high-frequency details of the experimental PSF across an extensive depth range--a challenging feat for alternative approaches. As an effective tool, VISPR enables the quantitative assessment of aberrations induced by the system and sample environment. From the simulations and experiments, we verified the superiority and effectiveness of VISPR. It is essential to highlight that VISPR applies to various SMLM microscope modalities.

molecular biology↗

bioRxiv2023

Melanophilin mediates the association of myosin-5a with melanosome via three distinct interactions

Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, i.e., the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes.

cell biology↗

bioRxiv2023

Healthy adults favor stable left/right hand choices over performance at an unconstrained reach-to-grasp task

Reach-to-grasp actions are fundamental to the daily activities of human life, but few methods exist to assess individuals reaching and grasping actions in unconstrained environments. The Block Building Task (BBT) provides an opportunity to directly observe and quantify these actions, including left/right hand choices. Here we sought to investigate the motor and non-motor causes of left/right hand choices, and optimize the design of the BBT, by manipulating motor and non-motor difficulty in the BBTs unconstrained reach-to-grasp task We hypothesized that greater motor and non-motor (e.g. cognitive/perceptual) difficulty would drive increased usage of the dominant hand. To test this hypothesis, we modulated block size (large vs. small) to influence motor difficulty, and model complexity (10 vs. 5 blocks per model) to influence non-motor difficulty, in healthy adults (n=57). We hypothesized that healthy adults with high non-dominant hand performance in a precision drawing task should be more likely to use their non-dominant hand in the BBT. Our data revealed that increased motor and non-motor difficulty led to lower task performance (slower speed), but participants only increased use of their dominant hand only under the most difficult combination of conditions: in other words, participants allowed their performance to degrade before changing hand choices, even though participants were instructed only to optimize performance. These results demonstrate that hand choices during reach-to grasp actions are more stable than motor performance in healthy right-handed adults, but tasks with multifaceted difficulties can drive individuals to rely more on their dominant hand. Statements and DeclarationsDr. Philip and Washington University in St. Louis have a licensing agreement with PlatformSTL to commercialize the iPad app used in this study.

neuroscience↗

bioRxiv2023

G protein subunit Gγ13-mediated signaling pathway is critical to the inflammation resolution and functional recovery of severely injured lungs

Tuft cells are a group of rare epithelial cells that can detect pathogenic microbes and parasites. Many of these cells express signaling proteins initially found in taste buds. It is, however, not well understood how these taste signaling proteins contribute to the response to the invading pathogens or to the recovery of injured tissues. In this study, we conditionally nullified the signaling G protein subunit G{gamma}13 and found that the number of ectopic tuft cells in the injured lung was reduced following the infection of the influenza virus H1N1. Furthermore, the infected mutant mice exhibited significantly larger areas of lung injury, increased macrophage infiltration, severer pulmonary epithelial leakage, augmented pyroptosis and cell death, greater bodyweight loss, slower recovery, worsened fibrosis and increased fatality. Our data demonstrate that the G{gamma}13-mediated signal transduction pathway is critical to tuft cells-mediated inflammation resolution and functional repair of the damaged lungs.To our best knowledge, it is the first report indicating subtype-specific contributions of tuft cells to the resolution and recovery.

immunology↗

bioRxiv2023

Extracellular vesicles from patients with coronary artery disease (CAD) demonstrate enhanced pro-coagulatory activity.

AimsExtracellular vesicles (EVs) carry unique repertoires of biologically active cargo that hold promise as novel biomarkers and future interventional targets for cardiovascular diseases (CVDs). However, it is unclear as to how the number, location, cellular origin, and size of these EVs within the circulation relate to the development of CVDs such as coronary artery disease (CAD). The current study compared these novel markers in arterial and venous blood samples in individuals undergoing coronary assessment with angiography.. EVs were then characterized from those presenting with and without CAD. Methods and ResultsArterial and venous blood samples were collected from individuals with confirmed CAD following coronary angiography and a matched cohort in whom there was no evidence of disease. EV fractions were isolated from 500{micro}L of platelet free plasma (PFP) by size exclusion chromatography. EVs were analyzed by Nanoparticle Tracking Analysis and flow cytometry to characterize number, size and cellular origin. A thrombin generation assay was used to assess the pro-coagulatory activity of the isolated circulating EVs. Proteomics was used to determine the protein cargo carried within the EVs. Coagulatory activity of EVs isolated from CAD patients was significantly higher in when compared to controls, although the numbers of EVs did not differ. There were higher numbers of endothelial-derived EVs in arterial blood compared with venous blood. Linear regression models revealed that plasma triacylglycerol concentration and age independently predicted circulating EV numbers in CAD patients. Proteomics revealed several proteins associated with coagulation upregulated in patients with CAD. ConclusionsAlthough the absolute numbers of EVs in CAD patients were not elevated, EVs in CAD patients had greater pro-coagulant activity, highlighting a potentially important role for EVs in the pathogenesis of CAD. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LICAD patients displayed a greater EV-induced thrombin generation capability when compated to those with no disease. C_LIO_LIThe proteomic profile of arterial-derived EVs in patients with CAD demonstrated several proteins associated with coagulation that may be a risk factor for developing a future event. C_LI What are the clinical implications?O_LIThe identification of EVs in patients with CAD with upregulated coagulation proteins may be a novel biomarker for the development of future events and may identify a high risk patient group that may benefit for more aggressive cardiovascular risk intervention. C_LI

physiology↗

bioRxiv2023

Connectome Operations For FSL ExEcution (COFFEE): a turnkey pipeline for preprocessing of fMRI data

Volumetric analysis methods continue to enjoy great popularity in the analysis of task-related functional MRI (fMRI) data. Among these methods, the software package FSL (FMRIB, Oxford, UK) is omnipresent throughout the field. However, it remains unknown what advantages might be gained by integrating FSL with alternative preprocessing tools. Here we developed the One-step General Registration and Extraction (OGRE) pipeline to apply FreeSurfer brain extraction for simultaneous registration and motion correction ("one-step resampling"), for FSL volumetric analysis of fMRI data. We compared three preprocessing approaches (OGRE, FSL, and fMRIPrep) in a dataset wherein adult human volunteers (N=39) performed a precision drawing task during fMRI scanning. The three approaches produced grossly similar results, but OGREs preprocessing led to lower inter-individual variability across the brain and greater detected activation in primary motor cortex contralateral to movement. This demonstrates that FreeSurfer tools and one-step resampling can improve FSLs volumetric analysis of fMRI data. The OGRE pipeline provides an off-the-shelf method to apply FreeSurfer-based brain extraction and one-step resampling of motion correction and registration for FSL analysis of task fMRI data.

neuroscience↗

bioRxiv2023

Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes

Alzheimers disease is characterized pathologically by cerebral deposition of 42-residue amyloid {beta}-peptide (A{beta}42), proteolytically produced from amyloid precursor protein (APP) by {beta}- and {gamma}-secretases.1 Although mutations in APP and presenilin, the catalytic component of {gamma}-secretase, cause familial Alzheimers disease (FAD), a role for A{beta}42 as the primary disease driver has not been clearly established and remains controversial.2,3 Here we show through comprehensive analysis of the multi-step proteolysis of APP substrate C99 by {gamma}-secretase that FAD mutations are consistently deficient in early proteolytic events, not later events that produce secreted A{beta} peptides. Cryo-electron microscopy revealed that a substrate mimetic traps {gamma}-secretase at the transition state for intramembrane proteolysis, and this structure closely aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and fluorescence lifetime imaging microscopy in cultured cells support stabilization by FAD mutations of enzyme-substrate and/or enzyme-intermediate complexes. Neuronal expression of C99 and/or presenilin-1 in Caenorabditis elegans led to age-dependent synaptic loss only when one of the transgenes carried an FAD mutation. Designed mutations that stabilize the enzyme-substrate complex and block proteolysis likewise led to synaptic loss. Collectively, these findings implicate the stalled process--not the released products--of {gamma}-secretase cleavage of substrates in FAD pathogenesis.

neuroscience↗