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Principal Investigator: Yihong Ye
Organization: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
Fiscal Year: 2023
Award: $779,674
Funding agency: National Institute of Diabetes and Digestive and Kidney Diseases
Proteinaceous inclusions termed Lewy bodies (LBs) are a classical hallmark of Parkinson's disease (PD). The primary component of these inclusions is alpha-synuclein (a-syn), a protein with an intrinsic propensity to misfold and aggregate. In PD patients, alpha-syn inclusions first observed in the olfactory bulb and the dorsal motor nucleus, progressively spread throughout the brain. Further findings that healthy embryonic dopamine neurons transplanted into PD patients developed LBs points suggest the tantalizing possibility of neuron-to-neuron transmission of a-syn. Subsequent work confirmed that synthetic a-syn pre-formed fibrils (PFFs) can be taken up by neurons, eliciting the misfolding of endogenous -syn into insoluble Lewy-like inclusions. Collectively, these studies led to the prion hypothesis of PD, wherein misfolded -syn provides a template for seeding new aggregates, propagating misfolded a-syn, and associated cytotoxicity. Thus, the propagation of alpha-syn is a viable new target to be explored in the development of new PD therapies.
The intercellular transmission of synuclein consists of two key steps: the secretion of synuclein from a donor neuron and its uptake by a recipient neuron. Misfolding-associated protein secretion (MAPS) is a recently discovered protein quality control process that selectively exports misfolded cytosolic proteins including a-syn. Secretion through MAPS requires the membrane-localized deubiquitinase USP19, which recruits aberrant polypeptides to the endoplasmic reticulum (ER) surface to facilitate their incorporation into late endosomes that are in tight association with the ER. Misfolded proteins are secreted to the extracellular milieu when late endosomes fuse with the plasma membrane.
The fate of the released misfolded proteins is currently unknown. Our recent studies suggest that mammalian cells can internalize misfolded proteins via endocytosis, but it is unclear whether they possess one or more receptors for misfolded proteins. Whether internalized proteins can impose damage prior to degradation by the lysosome is also unclear. The proposal is to elucidate the physiological relevance of the MAPS pathway by characterizing the interplay between secreted misfolded proteins and target cells.
Using a proximity-based ligation approach, we have used purified Tau as a bait to identify candidate Protein aggregates formed by microtubule-associated protein Tau can be transmitted in a stereotypic pattern in human brains, which correlates with the progression of several neurodegenerative diseases collectively termed Tauopathies. This process requires Tau, released from neurons, to interact with a cell surface receptor on a target cell, but little is known about the underlying mechanisms and the downstream pathophysiological consequences, particularly for Tau that engages glial cells. Using a spatially resolved proteomic mapping strategy, we identify integrin V/1, a heterodimer of the integrin family, as a receptor that not only mediates Tau fibril entry into astrocytes but also activates integrin signaling upon Tau binding. We show that distinct Tau species differentially activate integrin signaling, with filamentous Tau being a more robust stimulator. This leads to NFB activation and differential upregulation of pro-inflammatory cytokines and chemokines. Additionally, a sub-group of neurotoxic astrocyte markers are also induced in an integrin-dependent manner preferentially by filamentous Tau, causing astrocytes to release a neurotoxic factor(s). Together, these findings establish a paradigm that astrocytes can be directly converted into a neurotoxic state by filamentous Tau via an integrin receptor, which as a sensor transducing unfolded protein pathology, may provide a new therapeutic target for Tauopathies.
In a more recent effort, we used RNAseq to analyze the gene expression profile changes in response to a proteotoxic insult using tau fibrils as a model. We find that in addition to integrin signing, tau fibril ligation to an integrin receptor also activates PI3K, which leads to a cellular response that is distinct from the canonical integrin signaling. We further analyzed the secretome of cells treated with tau fibrils and found that many proteins are secreted in a tau-inducible manner. Among the secreted proteins, we identified C3 complement and Mmp3 metalloprotease as key regulators of neuronal cell death. This study reveals why astrocytes stimulated by tau fibrils can generate a pathological response via a physiologically critical cell surface receptor.
We recently reported that the infection of human cells by SARS-CoV-2, the virus underlying the ongoing COVID-19 pandemic depends on an endocytosis mechanism akin to the entry of alpha-Syn fibrils. Accordingly, knockdown of cell surface HSPG or cortex actin reduces viral infection in an in vitro cell-based model. A drug repurpose screen has identified several FDA-approved drugs that can effectively block the entry of SARS-CoV-2 in vitro. We further showed that in addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of Spike with a downstream receptor to promote viral entry. We reported that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a heparan sulfate-spike complex to compromise the fusogenic function of Spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar heparan sulfate-binding activities but with reduced affinity for DNA topoisomerases may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.
To identify additional viral entry inhibitors, we also developed and trained a graph convolutional network (GCN)-based classification model using information extracted from experimentally identified HSPG and actin inhibitors. This method allowed us to virtually screen 170,000 compounds, resulting in 2000 potential hits. A hit confirmation assay with the uptake of a fluorescence-labeled HSPG cargo further shortlisted 256 active compounds. Among them, 16 compounds had modest to the strong inhibitory activity. Interestingly, one of the compounds characterized binds heparan sulfate similarly as Mitoxantrone. In addition to inhibiting viral entry, it also diminished infection associated cell-cell fusion, which is often observed in severe COVID-10 patient. Mechanistically, this drug blocks spike-induced ACE2 clustering, which is required to enhance the fusion efficiency. We propose that drugs targeting heparan sulfate might be further developed to treat severe COVID-19.
Terms: <1-Phosphatidylinositol 3-Kinase><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><3-D><3-Dimensional><3D><ACE2><Actins><Affinity><Alternative Therapies><Assay><Astrocytes><Astrocytus><Astroglia><Binding><Bioassay><Biologic Assays><Biological Assay><Body Tissues><Brain><Brain Nervous System><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 S protein><COVID-19 crisis><COVID-19 epidemic><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 pandemic><COVID-19 public health crisis><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 virus><COVID19 S protein><COVID19 crisis><COVID19 epidemic><COVID19 global health crisis><COVID19 global pandemic><COVID19 health crisis><COVID19 pandemic><COVID19 public health crisis><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 virus><Cell Body><Cell Communication and Signaling><Cell Signaling><Cell Surface Receptors><Cell fusion><Cell membrane><Cell surface><Cells><Charge><Chemicals><Chemotactic Cytokines><Classification><Closure by Ligation><CoV-2><CoV2><Complement><Complement Proteins><Complex><Cytoplasmic Membrane><DA Neuron><DHAQ><DNA Topoisomerases><Degenerative Neurologic Diseases><Degenerative Neurologic Disorders><Development><Dihydroxyanthracenedione><Disease><Disorder><Dopamine neuron><Dorsal Motor Nucleus of the Vagus><Drug Targeting><Drugs><Embryo><Embryonic><Encephalon><Endocytosis><Endoplasmic Reticulum><Ergastoplasm><Expression Signature><FDA approved><Family><Fluorescence><Gene Expression Monitoring><Gene Expression Pattern Analysis><Gene Expression Profile><Gene Expression Profiling><Glia><Glial Cells><Glycosaminoglycans><Goals><Golgi><Golgi Apparatus><Golgi Complex><HSPG><Health><Heparan Sulfate><Heparan Sulfate Proteoglycan><Heparitin Sulfate><Homologous Chemotactic Cytokines><Human><In Vitro><Infection><Inflammatory><Integrins><Integrins Extracellular Matrix><Intercrines><Intracellular Communication and Signaling><Kolliker's reticulum><Label><Lewy Bodies><Ligation><Lung><Lung Respiratory System><Lysosomes><Lytotoxicity><MAPT gene><MAPT protein><MT-bound tau><MTBT1><Mammalian Cell><Maps><Mediating><Medication><Membrane><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Metallopeptidases><Metalloproteases><Metalloproteinases><Methods><Mitoxantrone><Mitozantrone><Modeling><Modern Man><Molecular Interaction><Mucopolysaccharides><NAC precursor><Nerve Cells><Nerve Impulse Transmission><Nerve Transmission><Nerve Unit><Nervous System Degenerative Diseases><Network-based><Neural Cell><Neural Degenerative Diseases><Neural degenerative Disorders><Neurocyte><Neurodegenerative Diseases><Neurodegenerative Disorders><Neuroglia><Neuroglial Cells><Neurologic Degenerative Conditions><Neuronal Transmission><Neurons><Non-neuronal cell><Nonneuronal cell><Nucleosomes><PARK1 protein><PARK4 protein><PI-3 Kinase><PI3-Kinase><PI3CG><PI3KGamma><PI3k><PIK3><PIK3CG><PIK3CG gene><Paralysis Agitans><Parkinson><Parkinson Disease><Pathologic><Pathology><Pathway interactions><Patients><Pattern><Peptide Signal Sequences><Pharmaceutic Preparations><Pharmaceutical Preparations><Phosphatidylinositol 3-Kinase><Phosphatidylinositol-3-OH Kinase><Phosphoinositide 3-Hydroxykinase><Physiologic><Physiological><Plasma Membrane><Primary Parkinsonism><Process><Protein Export><Protein Export Pathway><Protein Secretion><Proteins><Proteoheparan Sulfate><Proteomics><PtdIns 3-Kinase><Quality Control><R-Series Research Projects><R01 Mechanism><R01 Program><RNA Seq><RNA sequencing><RNAseq><Receptor Protein><Reporting><Research Grants><Research Project Grants><Research Projects><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 S protein><SARS-CoV-2 entry inhibitor><SARS-CoV-2 epidemic><SARS-CoV-2 global health crisis><SARS-CoV-2 global pandemic><SARS-CoV-2 pandemic><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 epidemic><SARS-CoV2 pandemic><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 epidemic><SARS-coronavirus-2 pandemic><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><SIS cytokines><SNCA><SNCA protein><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 epidemic><Severe Acute Respiratory Syndrome CoV 2 pandemic><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 entry inhibitor><Severe acute respiratory syndrome coronavirus 2 epidemic><Severe acute respiratory syndrome coronavirus 2 pandemic><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Signal Peptide><Signal Sequences><Signal Transduction><Signal Transduction Systems><Signaling><Stereotyping><Subgroup><Surface><Surface Proteins><System><Systematics><Tauopathies><Tissues><Topoisomerase Inhibitors><Toxic effect><Toxicities><Training><Transcript Expression Analyses><Transcript Expression Analysis><Transmission><Transplantation><Type I Phosphatidylinositol Kinase><Type III Phosphoinositide 3-Kinase><Up-Regulation><Upregulation><Vaccines><Viral><Viral Diseases><Virus><Virus Diseases><Work><Wuhan coronavirus><a-syn><a-synuclein><aberrant folded protein><aberrant folded proteins><abnormal folded protein><abnormal folded proteins><abnormally aggregated tau protein><alpha synuclein><alpha synuclein gene><alphaSP22><analog><analyze gene expression><angiotensin converting enzyme 2><angiotensin converting enzyme II><astrocytic glia><asyn><axon signaling><axon-glial signaling><axonal signaling><biological signal transduction><block SARS-CoV-2 entry><block severe acute respiratory syndrome coronavirus 2 entry><block viral entry><breakthrough infection><candidate identification><chemoattractant cytokine><chemokine><corona virus disease 2019 epidemic><corona virus disease 2019 pandemic><coronavirus disease 2019 S protein><coronavirus disease 2019 crisis><coronavirus disease 2019 epidemic><coronavirus disease 2019 global health crisis><coronavirus disease 2019 global pandemic><coronavirus disease 2019 health crisis><coronavirus disease 2019 pandemic><coronavirus disease 2019 public health crisis><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 virus><coronavirus disease crisis><coronavirus disease epidemic><coronavirus disease pandemic><coronavirus disease-19 global pandemic><coronavirus disease-19 pandemic><coronavirus disease-19 virus><cytokine><cytotoxicity><de-ubiquitinase><de-ubiquitinating enzyme><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><developmental><dopaminergic neuron><dorsal motor nucleus><drug repurposing><drug/agent><extracellular><filamentous tau inclusion><gene expression analysis><gene expression assay><gene expression pattern><gene expression signature><glia signaling><glial signaling><graph attention network><graph convolutional network><graph neural network><hCoV19><inhibit SARS-CoV-2 entry><inhibit severe acute respiratory syndrome coronavirus 2 entry><inhibit viral entry><inhibitor><insoluble aggregate><knock-down><knockdown><late endosome><life-threatening COVID><life-threatening COVID-19><life-threatening SARS-CoV-2><life-threatening coronavirus disease><life-threatening coronavirus disease 2019><life-threatening severe acute respiratory syndrome coronavirus 2><membrane structure><microtubule associated protein tau><microtubule associated protein tau aggregation><microtubule associated protein tau deposit><microtubule bound tau><microtubule-associated protein tau><microtubule-bound tau><misfolded protein><misfolded proteins><nCoV2><nerve cell death><nerve cell loss><nerve cement><nerve signaling><neural signaling><neurodegenerative illness><neuron cell death><neuron cell loss><neuron death><neuron loss><neuronal><neuronal cell death><neuronal cell loss><neuronal death><neuronal loss><neuronal signaling><neuropathologic tau><neuropathological tau><neurotoxic><neurotransmission><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><non A-beta component of AD amyloid><non A4 component of amyloid precursor><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><olfactory bulb><paired helical filament of tau><pathway><plasmalemma><polypeptide><pre-formed fibril><prion hypothesis><protein aggregate><protein aggregation><protein signal sequence><protein-only hypothesis><proteotoxic><proteotoxic protein><proteotoxicity><proteotoxin><pulmonary><receptor><recruit><repurposing agent><repurposing medication><response><self-aggregate tau><sensor><serious COVID><serious COVID-19><serious SARS-CoV-2><serious coronavirus disease><serious coronavirus disease 2019><serious severe acute respiratory syndrome coronavirus 2><severe COVID><severe COVID-19><severe COVID19><severe SARS-CoV-2><severe acute respiratory syndrome coronavirus 2 global health crisis><severe acute respiratory syndrome coronavirus 2 global pandemic><severe coronavirus disease><severe coronavirus disease 19><severe coronavirus disease 2019><severe severe acute respiratory syndrome coronavirus 2><synuclein><tau><tau PHF><tau Proteins><tau accumulation><tau aggregate><tau aggregation><tau associated neurodegeneration><tau associated neurodegenerative process><tau factor><tau fibrillization><tau filament><tau induced neurodegeneration><tau mediated neurodegeneration><tau neurodegenerative disease><tau neurofibrillary tangle><tau neuropathology><tau oligomer><tau paired helical filament><tau polymerization><tau-tau interaction><tauopathic neurodegenerative disorder><tauopathy><three dimensional><transcriptional profile><transcriptional profiling><transcriptional signature><transcriptome sequencing><transcriptomic sequencing><transmission process><transplant><ubiquitin isopeptidase><ubiquitin-specific isopeptidase><uptake><viral entry blocker><viral entry inhibitor><viral infection><virtual><virus infection><virus-induced disease><α synuclein gene><α-syn><α-synuclein><τ Proteins><τ aggregation>