Biology subjects

Pan, J.

Publications and source records attributed to Pan, J..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

SHP2-triggered endothelium activation fuels estradiol-independent endometrial sterile inflammation

Endometrial hyperplasia (EH) is a common gynecological disease primarily driven by excess estrogen. How endometrial sterile inflammation regulates EH remains unclear. First, we found the upregulation of SHP2 in endometrial endothelial cells from patients with EH by scRNA-Seq. SHP2 promoted inflammatory activation of endothelial cells, which promoted macrophage transendothelial migration. Subsequently, IL1{beta} and TNF from macrophages gave a feedforward loop to enhance endothelial cell activation and result in more IGF1 secretion, thereby sustaining sterile endometrial inflammation and facilitating endometrial epithelial cell proliferation even after estradiol withdrawal. Mechanistically, results of bulk RNA-Seq and phosphoproteomic analyses showed that endothelial SHP2 dephosphorylated RIPK1Y380 after estradiol stimulation. This event promoted activator protein 1 (AP-1) activation, instigating inflammation and increased CXCL10, CXCL13, COX2 and IGF1 secretion. Furthermore, targeting SHP2 by SHP099 or endothelial-specific SHP2 deletion alleviated EH progression in mice. Collectively, our findings demonstrate that SHP2 mediates the transition of endothelial activation, from estradiol-driven short inflammation to macrophage-amplified continuous sterile inflammation. Targeting chronic sterile inflammation mediated by endothelial cell activation is a promising strategy for non-hormonal intervention in EH.

molecular biology↗

bioRxiv2024

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

microbiology↗

bioRxiv2023

Comprehensive Multi-Omic Analysis Reveals Distinct Molecular Features in Early and Advanced Stages of Hepatocellular Carcinoma

Hepatocellular Carcinoma (HCC) is a serious primary solid tumor that is prevalent worldwide. Due to its high mortality rate, it is crucial to explore both early diagnosis and advanced treatment for HCC. In recent years, multi-omic approaches have emerged as promising tools to identify biomarkers and investigate molecular mechanisms of biological processes and diseases. In this study, we performed proteomics, phosphoproteomics, metabolomics, and lipidomics to reveal the molecular features of early- and advanced-stage HCC. The data obtained from these omics were analyzed separately and then integrated to provide a comprehensive understanding of the disease. The multi-omic results unveiled intricate biological pathways and interaction networks underlying the initiation and progression of HCC. Moreover, we proposed specific potential biomarker panels for both early- and advanced-stage HCC by overlapping our data with CPTAC database, and deduced novel insights and mechanisms related to HCC origination and development.

cancer biology↗

bioRxiv2023

Structural basis for VLDLR recognition by eastern equine encephalitis virus

Alphaviruses are arthropod-borne enveloped RNA viruses that include several important human pathogens with outbreak potential. Among them, eastern equine encephalitis virus (EEEV) is the most virulent, and many survivors develop neurological sequelae, including paralysis and intellectual disability. The spike proteins of alphaviruses comprise trimers of heterodimers of their envelope glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), two closely related proteins that are expressed in the brain, as cellular receptors for EEEV and a distantly related alphavirus, Semliki forest virus (SFV)1. The EEEV and SFV spike glycoproteins have low sequence homology, and how they have evolved to bind the same cellular receptors is unknown. Here, we used single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain. The structures reveal that EEEV and SFV use distinct surfaces to bind VLDLR; EEEV uses a cluster of basic residues on the E2 subunit of its spike glycoprotein, while SFV uses two basic residues at a remote site on its E1 glycoprotein. Our studies reveal that different alphaviruses interact with the same cellular receptor through divergent binding modes. They further suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.

microbiology↗

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

Spatially and Functionally Distinct mTORC1 Entities Orchestrate the Cellular Response to Amino Acid Availability

Amino acid (AA) availability is a robust determinant of cell growth, through controlling mTORC1 activity 1. According to the predominant model in the field, AA sufficiency drives the recruitment and activation of mTORC1 on the lysosomal surface by the heterodimeric Rag GTPases, from where it coordinates the majority of cellular processes (reviewed in 2,3). Importantly, however, 15 years after its initial discovery, the teleonomy of the proposed lysosomal regulation of mTORC1, and where mTORC1 acts on its effector proteins remain enigmatic 4. Here, by using multiple pharmacological and genetic means to perturb the lysosomal AA sensing and protein recycling machineries, we describe the spatial separation of mTORC1 regulation and downstream functions in mammalian cells, with lysosomal and non-lysosomal mTORC1 phosphorylating distinct substrates in response to different AA sources. Moreover, we reveal that a fraction of mTOR localizes at lysosomes due to basal lysosomal proteolysis that locally supplies new AAs, even in cells grown in the presence of extracellular nutrients, whereas cytoplasmic mTORC1 is regulated by exogenous AAs. Overall, our study substantially expands our knowledge about the topology of mTORC1 regulation by AAs, and hints at the existence of distinct, Rag- and lysosome-independent mechanisms that control its activity at other subcellular locations. Given the importance of mTORC1 signalling and AA sensing for human ageing and disease 2, our findings will likely open new directions toward the identification of function-specific mTORC1 regulators, and suggest new targets for drug discovery against conditions with dysregulated mTORC1 activity in the future.

cell biology↗

bioRxiv2023

A role for the ATP-dependent DNA ligase Lig E of Neisseria gonorrhoeae in biofilm formation

The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection characteristics. This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells which coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation, providing insight into the binding dynamics of these minimal DNA ligases.

microbiology↗

bioRxiv2023

Real-Time Spatiotemporal Measurement of Extracellular Signaling Molecules Using an Aptamer Switch-Conjugated Hydrogel Matrix

Cells rely on secreted signaling molecules to coordinate essential biological functions including development, metabolism, and immunity. Unfortunately, such signaling processes remain difficult to measure with sufficient chemical specificity and temporal resolution. To address this need, we have developed an aptamer-conjugated hydrogel matrix that enables continuous fluorescent measurement of specific secreted analytes - in two dimensions, in real-time. As a proof of concept, we performed real-time imaging of Dictyostelium discoideum cells, a well-studied amoeba model wherein inter-cellular communication is performed though cAMP signaling. We engineered a set of aptamer switches that generate a rapid and reversible change in fluorescence in response to cAMP signals. By combining multiple switches with different dynamic ranges, we can measure cAMP concentrations spanning three orders of magnitude in a single experiment. These sensors are embedded within a biocompatible hydrogel on which cells are cultured and their cAMP secretions can be imaged using fluorescent microscopy. Using this aptamer-hydrogel material system, we achieved the first direct measurements of oscillatory cAMP signaling that correlate closely with previous indirect measurements. Using different aptamer switches, this approach could be generalized for measuring other secreted molecules to directly visualize diverse extracellular signaling processes and the biological effects that they trigger in recipient cells.

bioengineering↗

bioRxiv2023

Integration of eQTL and Machine Learning Methods to Dissect Causal Genes with Pleiotropic effects in Genetic Regulation Networks of Seed Cotton Yield

Expression quantitative trait loci (eQTL) provide a powerful means of investigating the biological basis of genome-wide association study (GWAS) results and exploring complex traits or phenotypes. In addition to identifying the causal gene in cis, eQTL analysis also reveals a large number of trans-regulated genes located on different chromosomes, which form a gene regulatory network (GRN) that complements the GWAS locus. However, the dissection of a GRN and the crosstalk underlying multiple agronomical traits, along with prioritizing important genes in eQTL-derived GRNs, remains a major challenge. In this study, we generated 558 transcriptional profiles of lint-bearing ovules at one day post-anthesis (DPA) from a selective core cotton germplasm, from which we identified 12,207 eQTLs. By integrating with a GWAS catalog, we found that 66 out of 187 (35.29%) known phenotypic GWAS loci are colocalized with 1,090 eQTLs, forming 38 major functional GRNs predominantly (30 out of 38) associated with seed size-related phenotypes. Of the eGenes, 34 were shared between at least two functional GRNs, exhibiting pleiotropic effects, such as NF-YB3, GRDP1, and IDD7. Narrow-sense heritability analysis showed that the heritability increased with combining the eQTLs with GRNs compared to those with previous yield trait GWAS loci. The extreme gradient boosting (XGBoost) machine learning approach was then applied to predict seed cotton yield phenotypes based on gene expression. Top-ranking eGenes (NF-YB3, FLA2, and GRDP1) derived by XGBoost with pleiotropic effects on yield traits were validated, along with their potential roles by correlation analysis, domestication selection analysis, and transgenic plants. This study provides insights into the mining of GRNs in relation to the pleiotropy of phenotype. The combination of eQTL and machine learning approaches is efficient in improving the genetic dissection of agricultural traits.

genetics↗

bioRxiv2023

Spatiotemporal transcriptome atlas of human embryos after gastrulation

The spatial and temporal atlas of gene expression in the human embryo at early gestation is critical in understanding embryo development, organogenesis, and disease origins. We obtained the spatiotemporal transcriptome from 90 sagittal sections of 16 whole human embryos from 3 to 8 post-conception weeks by Stereo-seq with high resolution and ultra-large field, establishing the development trajectory/regulatory profiling of 49 organs. We uncovered the organ-specific regulons as potential lineage-determining factors and identified the new regulatory networks during heart and brain development. The atlas refines the key organs/cell types vulnerable to virus infection and genetic disorders, and, reveals the dynamics of allelic gene expression in specific organs at different stages. These results present the first comprehensive delineation of the spatiotemporal transcriptomic dynamics of human organogenesis. One Sentence SummaryThe spatiotemporal transcriptome atlas presents a comprehensive delineation of human embryogenesis after gastrulation.

developmental biology↗

bioRxiv2023

A rapid ELISA platform with no sample preparation requirement

Since its invention in the 1970s, the enzyme-linked immunosorbent assay (ELISA) has served as the "gold-standard" for blood and plasma protein biomarker quantification. However, ELISAs require significant amounts of sample preparation entailing multiple reagent additions, incubations, and washing steps, limiting their clinical usefulness in the context of diagnosis and prognosis of rapidly evolving medical conditions. In this work, we describe the instant ELISA biosensor platform, a probe that can be exposed directly to blood or other biological samples and quantifies protein biomarkers within 15 minutes. The sensor leverages a novel affinity reagent termed monolithic dual-antibody clamp (MDAC) which preserves the specificity, sensitivity, and generalizability of ELISA while also enabling rapid analysis of unprocessed blood and other complex matrices. Using MDAC in chicken media, we demonstrate picomolar quantification of the inflammatory marker tumor necrosis factor alpha (TNF), as well as monocyte chemotactic protein (MCP)-1, a useful prognostic indicator of cytokine release syndrome (CRS) during chimeric antigen receptor (CAR) T-cell immunotherapy. Finally, we demonstrate MCP-1 quantification in plasma samples from patients who had undergone CAR T-cell treatment.

bioengineering↗

bioRxiv2023

Clathrin Light Chains are essential in negative regulation of cell death and immunity in Arabidopsis through interacting with autophagy pathway

Clathrin plays a critical role in clathrin-mediated endocytosis (CME) in plants, and it is required for autophagy in mammals. However, the functional interconnection of clathrin with autophagy has not been firmly established in plants. Here, we demonstrate that loss of function of clathrin light chain (CLC) subunit 2 and 3 results in salicylic acid (SA)- and H2O2-dependent accelerated senescence and activated defense responses in Arabidopsis, which are hallmarks of the autophagy-related gene (ATG) mutants. Similar to atg mutants, the clc2-1clc3-1 double mutant has enhanced sensitivity to both carbon and nitrogen starvation and enhanced resistance to biotrophic bacterial and fungal pathogens. In addition, the autophagy flux was significantly reduced in the roots of clc2-1clc3-1 mutant plants relative to Col-0 plants under carbon starvation conditions. Furthermore, our Yeast-2-hybrid (Y2H) and Luciferase complementation assays showed that CLC2 directly interacted with ATG8h and ATG8i. Mutations within the unique ATG8-interacting motif (AIM) of CLC2 as well as at the LIR/AIM-docking site (LDS) of ATG8h abolished the interaction between CLC2 and ATG8h. As anticipated, both GFP-ATG8h/GFP-ATG8i and CLC2 were subjected to autophagic degradation in the vacuoles. Together, our data revealed that the accelerated senescence and activated immune responses observed in Arabidopsis clc2-1clc3-1 mutant plants result from impaired autophagy, and CLC2 participates in autophagy through direct interactions with ATG8h and ATG8i in an AIM1- and LDS-dependent manner. Our results unveil a previously unidentified link between the function of CLCs and autophagy.

plant biology↗

bioRxiv2023

Inhibition of type III secretion system induced leukotriene B4 production by Yersinia pestis: A mechanism for early immune evasion

Subverting the host immune response to inhibit inflammation is a key virulence factor of Yersinia pestis. The inflammatory cascade is tightly controlled via the sequential action of lipid and protein mediators of inflammation. Because delayed inflammation is essential for Y. pestis to cause lethal infection, defining the mechanisms used by Y. pestis to manipulate the inflammatory cascade is necessary to understand this pathogens virulence. While previous studies have established that Y. pestis actively inhibits the expression of host proteins that mediate inflammation, there is currently a gap in our understanding of inflammatory lipid mediator response during plague. Here we use in vivo lipidomics to define the synthesis of lipid mediators of inflammation within the lungs during pneumonic plague. Interestingly, while we observed an early cyclooxygenase response during pneumonic plague, there was a significant delay in the synthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid chemoattractant and activator of immune cells. Furthermore, in vitro studies with primary leukocytes from mice and humans further revealed that Y. pestis actively inhibited the synthesis of LTB4. Finally, using Y. pestis mutants in the Ysc type 3 secretion system (T3SS) and Yersinia outer protein (Yop) effectors, we demonstrate that leukocytes recognize the T3SS to initiate the synthesis of LTB4 rapidly. However, the Yop effectors secreted through the same system effectively inhibit this host response. Together, these data demonstrate that Y. pestis actively inhibits the synthesis of LTB4, an inflammatory lipid, required for rapid recruitment of leukocytes to the site of infection. Author SummaryYersinia pestis, the bacteria that causes plague, targets the hosts innate immune response to inhibit inflammation. Because the generation of this non-inflammatory environment is required for infection, we are interested in mechanisms used by Y. pestis to block inflammation. Lipid mediators are potent signaling molecules that regulate multiple host immune responses, including inflammation. While there have been studies on how Y. pestis blocks the proteins that mediate inflammation, there is a gap in our understanding of the inflammatory lipid mediator response during plague. Here we show that Y. pestis inhibits the production of one of these critical lipid mediators, leukotriene B4, by host immune cells. Furthermore, we identify both the signals that induce LTB4 production by leukocytes and the mechanisms used by Y. pestis to inhibit this process. Together, these data represent the first comprehensive analysis of inflammatory lipids produced during plague and improve our current understanding of how Y. pestis manipulates the host immune response to generate a permissive non-inflammatory environment required for bacterial colonization.

microbiology↗

bioRxiv2023

An Antibody-Based Molecular Switch for Continuous Biosensing

We present a generalizable approach for designing biosensors that can continuously detect specific biomarkers in real time and without sample preparation. This is achieved by converting existing antibodies into target-responsive "antibody-switches" that enable continuous optical biosensing. To engineer these switches, antibodies are linked to a molecular competitor through a DNA scaffold, such that competitive target binding induces scaffold switching and fluorescent signaling of changing target concentrations. As a demonstration, we designed antibody-switches that achieve rapid, sample-preparation-free sensing of digoxigenin and cortisol in undiluted plasma. We showed that, by substituting the molecular competitor, we can further modulate the sensitivity of our cortisol switch to achieve detection at concentrations spanning 3.3 nM to 3.3 mM. Finally, we integrated this switch with a fiber-optic sensor to achieve hours-long continuous sensing of cortisol in buffer with <5-minute time resolution. We believe this modular sensor design can enable continuous biosensor development for many biomarkers.

bioengineering↗

bioRxiv2023

E2F1 Mediates SOX17 Deficiency-Induced Pulmonary Hypertension

RationaleRare genetic variants and genetic variation at loci in an enhancer in SRY-Box Transcription Factor 17 (SOX17) are identified in patients with idiopathic pulmonary arterial hypertension (PAH) and PAH with congenital heart disease. However, the exact role of genetic variants or mutation in SOX17 in PAH pathogenesis has not been reported. ObjectivesTo investigate the role of SOX17 deficiency in pulmonary hypertension (PH) development. MethodsHuman lung tissue and endothelial cells (ECs) from IPAH patients were used to determine the expression of SOX17. Tie2Cre-mediated and EC-specific deletion of Sox17 mice were assessed for PH development. Single-cell RNA sequencing analysis, human lung ECs, and smooth muscle cell culture were performed to determine the role and mechanisms of SOX17 deficiency. A pharmacological approach was used in Sox17 deficiency mice for therapeutic implication. Measurement and Main ResultsSOX17 expression was downregulated in the lungs and pulmonary ECs of IPAH patients. Mice with Tie2Cre mediated Sox17 knockdown and EC-specific Sox17 deletion developed spontaneously mild PH. Loss of endothelial Sox17 in EC exacerbated hypoxia-induced PH in mice. Loss of SOX17 in lung ECs induced endothelial dysfunctions including upregulation of cell cycle programming, proliferative and anti-apoptotic phenotypes, augmentation of paracrine effect on pulmonary arterial smooth muscle cells, impaired cellular junction, and BMP signaling. E2F Transcription Factor 1 (E2F1) signaling was shown to mediate the SOX17 deficiency-induced EC dysfunction and PH development. ConclusionsOur study demonstrated that endothelial SOX17 deficiency induces PH through E2F1 and targeting E2F1 signaling represents a promising approach in PAH patients.

physiology↗

bioRxiv2023

A novel chimeric RNA originating from BmCPV S4 and Bombyx mori HDAC11 transcripts regulates virus proliferation

Polymerases encoded by segmented negative-strand RNA viruses cleave 5-m7G-capped host transcripts to prime viral mRNA synthesis ( cap-snatching) to generate chimeric RNA, and trans-splicing occurs between viral and cellular transcripts. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an RNA virus belonging to Reoviridae, is a major pathogen of silkworm (B. mori). The genome of BmCPV consists of 10 segmented double-stranded RNAs (S1-S10) from which viral RNAs encoding a protein are transcribed. In this study, chimeric silkworm-BmCPV RNAs, in which the sequence derived from the silkworm transcript could fuse with both the 5 end and the 3 end of viral RNA, were identified in the midgut of BmCPV-infected silkworms by RNA_seq and further confirmed by PCR and Sanger sequencing. A novel chimeric RNA, HDAC11-S4 RNA 4, derived from silkworm histone deacetylase 11 (HDAC11) and the BmCPV S4 transcript encoding viral structural protein 4 (VP4), was selected for validation by in situ hybridization and Northern blotting. Interestingly, our results indicated that HDAC11-S4 RNA 4 was generated in a BmCPV RNA-dependent RNA polymerase (RdRp)-independent manner and could be translated into a truncated BmCPV VP4 with a silkworm HDAC11-derived N-terminal extension. Moreover, it was confirmed that HDAC11-S4 RNA 4 inhibited BmCPV proliferation, decreased the level of H3K9me3 and increased the level of H3K9ac. These results indicated that during infection with BmCPV, a novel mechanism, different from that described in previous reports, allows the genesis of chimeric silkworm-BmCPV RNAs with biological functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/527451v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1b3b949org.highwire.dtl.DTLVardef@4972c0org.highwire.dtl.DTLVardef@cc15aorg.highwire.dtl.DTLVardef@16a0a19_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Astrocyte and neuronal Panx1 support long-term reference memory in mice

Pannexin 1 (Panx1) are ubiquitously expressed proteins that form plasma membrane channels permeable to anions and moderate sized signaling molecules (e.g., ATP, glutamate). In the nervous system, activation of Panx1 channels have been extensively shown to contribute to distinct neurological disorders (epilepsy, chronic pain, migraine, neuroAIDS, etc.) but knowledge of extent to which these channels have a physiological role remains restricted to three studies supporting their involvement in hippocampus dependent learning. Given that Panx1 channels may provide an important mechanism for activity-dependent neuron-glia interaction, we used Panx1 transgenic mice with global and cell-type specific deletions of Panx1 to interrogate their participation in working and reference memory. Using the 8-arm radial maze, we show that long-term spatial reference memory, but not spatial working memory, is deficient in Panx1-null mice and that both astrocyte and neuronal Panx1 contribute to the consolidation of long-term spatial memory. Field potential recordings in hippocampal slices of Panx1-null mice revealed an attenuation of both long-term potentiation (LTP) of synaptic strength and long-term depression (LTD) at Schaffer collateral - CA1 synapses without alterations basal synaptic transmission or pre-synaptic paired-pulse facilitation. Our results implicate both neuronal and astrocyte Panx1 channels as critical players for the development and maintenance of long-term spatial reference memory in mice.

neuroscience↗

bioRxiv2023

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies. Brief SummaryA mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.

cell biology↗