Biology subjects

Leng, K.

Publications and source records attributed to Leng, K..

14 recordsLinked to original sources

bioRxiv2026

Resilience to neuronal hyperactivity and restoration of the neuroimmune interactome by blocking fibrin-induced microglia activation in Alzheimers disease

Cerebrovascular pathology and neuronal network dysfunction are early features of Alzheimers disease (AD) associated with neuroinflammation and cognitive decline, but the vascular and immune triggers of neuronal hyperactivity remain largely unknown. Here, we show that the blood coagulation protein fibrin disrupts microglia-neuron interactions, promoting neuronal hyperactivity in an AD mouse model. Genetic elimination of the fibrin inflammatory domain reduced neuronal hyperactivity, restored dynamic microglial interactions with active neurons and protected from high-risk decision making in 5XFAD mice. Leveraging the transcriptional signatures of microglia and inhibitory and excitatory neurons, a ligand-receptor atlas revealed fibrin-dependent disruption of innate immune and glutamatergic signaling between microglia and neurons in AD mice. Patients with AD also showed a correlation of cerebrospinal fluid (CSF) fibrinogen levels with biomarkers of inflammation, vascular and synaptic dysfunction. Thus, resilience to neuronal hyperactivity and restoration of the neuroimmune interactome by targeting fibrin may have therapeutic implications for Alzheimers disease and related conditions. There is a companion manuscript submitted to bioRxiv (Lauderdale et al., 2026) HighlightsO_LIVascular-microglia axis drives neuronal hyperactivity C_LIO_LIFibrin inflammatory activity disrupts the microglia-neuron interactome C_LIO_LIMicroglia activation by fibrin impairs decision-making in AD mice C_LIO_LISynaptic dysfunction and immune biomarkers correlate with CSF fibrinogen in AD patients C_LI

neuroscience↗

bioRxiv2025

Increased Synapse Elimination by Inflammatory Cells Contributes to Long-lasting Post-Stroke Memory Dysfunction in Old Mice

Old patients are more likely to experience memory dysfunction than young patients after a stroke. It has been reported that brain astrocytes and microglia cause excessive removal of synapses at the acute and subacute stages of stroke, and inhibition of their phagocytosis improved neurobehavioral outcomes. We hypothesized that memory dysfunction in old subjects is associated with increased synapse removal by inflammatory cells. Ischemic stroke was induced in young (2-month-old) and old (15-18-month-old) mice. Memory functions were analyzed by the Y-maze test weekly for 8 weeks and the novel object recognition (NOR) test at 7 days before and 8 weeks post-stroke. We have also created a tibia fracture 6 hours before stroke injury in young mice, to test if the activation of 7-nicotinic acetylcholine receptor (nAchRs) reduces inflammatory cells and synapse elimination. Brains were collected 8 weeks after the induction of ischemic stroke. Transcriptome changes, neuronal injuries, neuroinflammation, synapse removal, and neurite outgrowth were analyzed. We found that old mice developed long-term memory dysfunction after ischemic stroke, which was not seen in young mice. Old mice showed larger infarct volume, higher neuroinflammation, and more synapses engulfed by microglia/macrophages and astrocytes in the peri-atrophic region and hippocampi than young mice. More synapse-engulfing astrocytes than microglia/macrophages were present in the peri-atrophic region and the ipsilateral hippocampi, suggesting that reactive astrocytes contributed more than activated microglia/macrophages in synapse removal. Activation of 7-nAchRs in mice subjected to tibia fracture 6 hours before ischemic injury reduced synapse removal by microglia/macrophages and astrocytes in the hippocampi. Our study indicated that an increase in synaptic elements by inflammatory cells contributes to the long-lasting memory deficit after stroke in old mice. Astrocytes may contribute more than microglia/macrophages in synapse removal. Inhibition of neuroinflammation by activating 7-nAchRs can reduce synapse loss and thus may improve post-stroke memory function.

neuroscience↗

bioRxiv2025

Regulators of Interferon-Responsive Microglia Uncovered by Genome-wide CRISPRi Screening

Microglia dynamically support brain homeostasis through the induction of specialized activation programs or states. One such program is the Interferon-Responsive Microglia state (IRM), which has been identified in developmental windows, aging, and disease. While the functional importance of this state is becoming increasingly clear, our understanding of the regulatory networks that govern IRM induction remain incomplete. To systematically identify genetic regulators of the IRM state, we conducted a genome-wide CRISPR interference (CRISPRi) screen in human iPSC-derived microglia (iPS-Microglia) using IFIT1 as a representative IRM marker. We identified 772 genes that modulate IRM, including canonical type I interferon signaling genes (IFNAR2, TYK2, STAT1/2, USP18) and novel regulators. We uncovered a non-canonical role for the CCR4-NOT complex subunit CNOT10 in IRM activation, independent of its traditional function. This work provides a comprehensive resource for dissecting IRM biology and highlights both established and novel targets for modulating microglial interferon signaling in health and disease.

neuroscience↗

bioRxiv2024

CRISPRi-based screen of Autism Spectrum Disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and uptake of synaptic material

Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified ADNP, a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.

neuroscience↗

bioRxiv2023

Cell type specificity of mosaic chromosome 1q gain resolved by snRNA-seq in a case of epilepsy with hyaline protoplasmic astrocytopathy

STRUCTURED ABSTRACTO_ST_ABSIntroductionC_ST_ABSMosaic gain of chromosome 1q (chr1q) has been associated with malformation of cortical development (MCD) and epilepsy. Hyaline protoplasmic astrocytopathy (HPA) is a rare neuropathological finding seen in cases of epilepsy with MCD. The cell-type specificity of mosaic chr1q gain in the brain and the molecular signatures of HPA are unknown. MethodsWe present a child with pharmacoresistant epilepsy who underwent epileptic focus resections at age 3 and 5 years and was found to have mosaic chr1q gain and HPA. We performed single-nuclei RNA-sequencing (snRNA-seq) of brain tissue from the second resection. ResultssnRNA-seq showed increased expression of chr1q genes specifically in subsets of neurons and astrocytes. Differentially expressed genes associated with inferred chr1q gain included AKT3 and genes associated with cell adhesion or migration. A subpopulation of astrocytes demonstrated marked enrichment for synapse-associated transcripts, possibly linked to the astrocytic inclusions observed in HPA. DiscussionsnRNA-seq may be used to infer the cell type-specificity of mosaic chromosomal copy number changes and identify associated gene expression alterations, which in the case of chr1q gain may involve aberrations in cell migration. Future studies using spatial profiling could yield further insights on the molecular signatures of HPA.

neuroscience↗

bioRxiv2022

Mechanistic implications of the interaction of the soluble substrate-binding protein with a type II ABC importer

ATP-Binding Cassette (ABC) Transporters employ homologous ATPase domains to drive transmembrane transport of diverse substrates ranging from small molecules to large polymers. Bacterial ABC importers require an extramembranous substrate binding protein (SBP) to deliver the transport substrate to the extracellular side of the transporter complex. Previous studies suggest significant differences in the transport mechanisms of type I vs. type II bacterial ABC importers, which contain unrelated transmembrane domains. We herein use ensemble fluorescence resonance energy transfer (FRET) experiments to characterize the kinetics of SBP interaction in the E. coli BtuCD-F complex, a canonical type II ABC importer that transports vitamin B12. We demonstrate that, in the absence of B12, BtuF (the SBP) forms a locked (kinetically hyper-stable) complex with nanodisc-reconstituted BtuCD that can only be dissociated by ATP hydrolysis, which represents a futile reaction cycle. Notably, no type I importer has been observed to form an equivalent locked complex. We also show that either ATP or vitamin B12 binding substantially slows formation of the locked BtuCD-F complex, which will limit the occurrence of futile hydrolysis under physiological conditions. Mutagenesis experiments demonstrate that efficient locking requires concerted interaction of BtuCD with residues on both sides of the B12 binding pocket in BtuF. Combined with the kinetic inhibition of locking by ATP binding, these observations imply that the transition state for the locking reaction involves a global alteration in the conformation of BtuCD that extends from its BtuF binding site in the periplasm to its ATP-binding sites on the opposite side of the membrane in the cytoplasm. These observations suggest that locking, which seals the extracellular B12 entry site of the transporter, may help push B12 through the transporter and directly contribute to the transport mechanism in type II ABC importers.

biophysics↗

bioRxiv2022

Realtime observation of ATP-driven single B12 molecule translocationthrough BtuCD-F

ATP-Binding Cassette (ABC) Transporters use ATP binding and hydrolysis to power transmembrane transport of chemically diverse substrates. Current knowledge of their mechanism comes primarily from static structures of stable intermediates along the transport cycle. Recently, single-molecule fluorescence resonance energy transfer (smFRET) measurements have generated insight into the functional dynamics of transmembrane transporters, but studies to date lack direct information on the physical movement of the transport substrate. Here, we report development of an smFRET system that exploits fluorescence quenching by vitamin B12 to track its location in real time during ATP-driven transport by nanodisc-reconstituted E. coli BtuCD-F, an extensively studied type II ABC importer. Our data demonstrate that transmembrane translocation of B12 is driven by two sequential high-energy conformational changes that are inaccessible to standard structural methods because they are inherently transient. The first moves B12 from the periplasm into the transmembrane domain of the transporter; notably, this reaction is driven by hydrolysis of a single ATP molecule, in contrast to the mechanism established for several other ABC Transporter families in which ATP-binding drives the mechanochemical power-stroke prior to hydrolysis. The second mediates B12 release on the opposite side of the transporter, and it is driven by formation of a hyper-stable complex between BtuCD and BtuF. Hydrolysis of a second single ATP molecule is then required to dissociate BtuCD from the BtuF substrate-binding protein to enable it to bind B12 and initiate another round of transport. Our experiments have visualized substrate translocation in real-time at a single-molecule level and provided unprecedented information on the mechanism and dynamics of a paradigmatic transmembrane transport process.

biophysics↗

bioRxiv2022

Granulin Loss of Function in Human Mature Brain Organoids Implicates Astrocytes in TDP-43 Pathology

Loss of function (LoF) of Tar-binding protein 43 (TDP-43) and mislocalization, together with TDP-43-positive and hyperphosphorylated inclusions, are found in postmortem tissue of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, including those carrying LoF variants in the progranulin gene (GRN). Modelling TDP-43 pathology has been challenging in vivo and in vitro. We present a 3D-induced pluripotent stem cell (iPSC)-derived paradigm - mature brain organoids (mbOrg) - composed of cortical-like-astrocytes (iA) and neurons (iN). When devoid of GRN, mbOrgs spontaneously recapitulate TDP-43 mislocalization, hyperphosphorylation and LoF phenotypes. Mixing-and-matching genotypes in mbOrgs showed that GRN-/- iA are drivers for TDP-43 pathology. Finally, we rescued TDP-43 LoF by adding exogenous progranulin, demonstrating a link between TDP-43 LoF and progranulin expression. In conclusion, we present an iPSC-derived platform that shows striking features of human TDP-43 proteinopathy and provides a tool for mechanistic modelling of TDP-43 pathology and patient-tailored therapeutic screening for FTD and ALS. HighlightsO_LIGRN-/- iPSC-derived 3D paradigm (mbOrg) composed of mature cortical-like astrocytes and neurons recapitulates features of TDP-43 proteinopathy C_LIO_LIGRN-/- cortical-like astrocytes are necessary and sufficient for the development of the TDP-43 loss of function phenotype in mbOrg. C_LIO_LIA TDP-43 phenotype can be rescued in neurons by treating neuron and astrocyte co-cultures with progranulin full length protein. C_LI eTOC blurbIn this article, de Majo and colleagues present a novel 3D iPSC-derived model to study neurodegenerative disorders such as ALS and FTD. When devoid of GRN expression, these cultures present features of ALS and FTD associated pathology hardly ever observed in vitro. These phenotypes are shown to be primarily driven by diseased astrocytes and can be rescued by progranulin supplementation.

neuroscience↗

bioRxiv2022

Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we rigorously compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides the best balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

genomics↗

bioRxiv2022

Reactive astrocytes transduce blood-brain barrier dysfunction through a TNFα-STAT3 signaling axis and secretion of alpha 1-antichymotrypsin

Astrocytes are critical components of the neurovascular unit that support blood-brain barrier (BBB) function in brain microvascular endothelial cells (BMECs). Transformation of astrocytes to a reactive state in response to injury and disease can be protective or harmful to BBB function, but the underlying mechanisms for these effects remain mostly unclear. Using a human induced pluripotent stem cell (iPSC)-derived coculture model of BMEC-like cells and astrocytes, we found that tumor necrosis factor alpha (TNF) transitions astrocytes to an inflammatory reactive state through activated STAT3 signaling, whereby the resultant astrocytes disrupt passive BBB function and induce vascular cell adhesion molecule 1 (VCAM-1) expression in the BMEC-like cells. These associations between inflammatory reactive astrocytes, STAT3 activation, and vascular VCAM-1 expression were corroborated in human postmortem tissue. Bioinformatic analyses coupled with CRISPR interference techniques in the iPSC model revealed that inflammatory reactive astrocytes transduce BBB disruption in part through SERPINA3, which encodes alpha 1-antichymotrypsin (1ACT), a secreted serine protease inhibitor associated with aging, neuroinflammation, and Alzheimers disease. In murine ex vivo cortical explant cultures, shRNA-mediated silencing of Serpina3n in astrocytes reduced vascular VCAM-1 expression after TNF challenge. Further, direct treatment with recombinant Serpina3n in both ex vivo explant cultures and the brain in vivo (via intracerebroventricular injection into wild-type mice) was sufficient to induce vascular VCAM-1 expression and reduce tight junction integrity. Overall, our results define the TNF-STAT3 signaling axis as a driver of an inflammatory reactive astrocyte subtype responsible for BBB dysfunction. Our results also identify 1ACT as an explicit mediator of BBB damage and suggest that inhibition of 1ACT expression or activity could represent a therapeutic avenue for reversing BBB deficits in aging and neurodegenerative disease.

neuroscience↗

bioRxiv2021

mTOR controls neurotoxic lysosome exocytosis in inflammatory reactive astrocytes

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo - IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states, was upregulated in astrocytes in multiple sclerosis lesions, and preferentially co-localized with astrocytes in hypoxic-ischemic encephalopathy. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

cell biology↗

bioRxiv2021

CRISPRi screens in human astrocytes elucidate regulators of distinct inflammatory reactive states

In response to central nervous system injury or disease, astrocytes become reactive, adopting context-dependent states and functional outputs. Certain inflammatory insults induce reactive astrocytes that lose homeostatic functions and gain harmful outputs through cellular pathways that are not fully understood. Here, we combined single-cell transcriptomics with CRISPRi screening in human iPSC-derived astrocytes to systematically interrogate inflammatory astrocyte reactivity. We found that autocrine-paracrine IL-6 and interferon signaling downstream of canonical NF-{kappa}B activation drove two distinct inflammatory reactive signatures - one promoted by and the other inhibited by STAT3. These signatures overlapped with those observed in other experimental contexts, including mouse models, and their markers were upregulated in the human brain in Alzheimers disease and hypoxic ischemic encephalopathy. Furthermore, we validated that these signatures were regulated by Stat3 in vivo. These results and the platform we established have the potential to guide the development of therapeutics to selectively modulate different aspects of inflammatory astrocyte reactivity.

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↗

bioRxiv2020

Molecular characterization of selectively vulnerable neurons in Alzheimer's Disease

Alzheimers disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus - brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively - from postmortem brains spanning the progression of AD-type tau neurofibrillary pathology. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex, and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience.

neuroscience↗