Biology subjects

Zhang, Y.-J.

Publications and source records attributed to Zhang, Y.-J..

18 recordsLinked to original sources

bioRxiv2026

Hepatitis C Virus Remodels Lipid Droplets to Promote Mitochondrial Fatty Acid Accumulation and Metabolic Activation

Hepatitis C virus (HCV) depends on host lipid metabolism and lipid droplets (LDs) for genome replication, assembly, and particle production, yet how LD structure and lipid utilization change over the course of infection remains incompletely understood. Here, we investigated the temporal remodeling of LD-associated metabolic pathways during HCV JFH-1 infection of human hepatoma Huh7 cells. HCV infection transiently increased LD fluorescence intensity at 24 hours post-infection (hpi), followed by normalization or relative loss of LD signal at later time points. Concomitantly, LDs became progressively clustered and enlargement during late infection, despite reduced protein levels of the canonical LD fusion proteins CIDEA, CIDEB, and CIDEC, suggesting that HCV-induced LD enlargement occurs through CIDE-independent mechanisms. Transcriptomic, RT-qPCR, and immunoblot analyses revealed time-dependent regulation of genes and proteins involved in LD structure, triglyceride synthesis, lipolysis, lipid uptake, and mitochondrial fatty acid utilization. Subcellular fractionation demonstrated preferential accumulation of fatty acids in mitochondrial fractions at 24-72 hpi. This redistribution was accompanied by increased oxygen consumption rate, elevated extracellular acidification, and progressive reactive oxygen species accumulation, indicating infection-associated metabolic activation and oxidative stress. Pharmacological inhibition of DGAT1-dependent LD biogenesis, LIPA-dependent lysosomal lipid hydrolysis, LIPE/HSL-dependent lipolysis, or CPT1-dependent mitochondrial fatty acid transport markedly reduced mitochondrial fatty acid accumulation and suppressed HCV-induced respiratory activity. Inhibition of LIPA or LIPE/HSL reduced both HCV RNA and core protein levels, whereas inhibition of CPT1 or DGAT1 had more pronounced effects on core protein than on viral RNA. Together, these findings support a model in which HCV dynamically remodels LDs, mobilizes LD-associated fatty acids, and redirects them toward mitochondria to support infection-associated metabolism and downstream stages of the viral life cycle. Lipid hydrolysis and mitochondrial fatty acid trafficking therefore represent potential host-directed targets for limiting HCV infection. SIGNIFIGANCEHepatitis C virus depends on host lipid metabolism for replication, assembly, and production of infectious particles, but how it uses lipid droplets over time remains incompletely understood. This study shows that hepatitis C virus dynamically remodels lipid droplets, causing an early increase in lipid storage followed by droplet enlargement and mobilization of fatty acids during later infection. The released fatty acids preferentially accumulate in mitochondria, where they are associated with increased cellular respiration and oxidative stress. Blocking lipid droplet formation, lipid breakdown, or fatty acid transport to mitochondria reduced this metabolic response and decreased viral RNA or core protein accumulation. Inhibition of lysosomal acid lipase and hormone-sensitive lipase suppressed both viral RNA and protein levels. These findings identify lipid droplet breakdown and mitochondrial fatty acid trafficking as important host processes used by hepatitis C virus and as potential targets for host-directed antiviral intervention.

microbiology↗

bioRxiv2026

Isolation of Zika Virus Replication Complex Reveals a Proviral Nuclear Factor

Zika virus (ZIKV) is an arthropod-borne flavivirus of international public health impact. ZIKV has a positive-sense, single-stranded RNA genome and remodels intracellular membranes to form replication complexes (RCs). The objective of this study was to isolate and characterize the RCs from ZIKV-infected cells and to identify host-cell components recruited to participate in viral replication. Here, we isolated the RCs from ZIKV-infected Vero cells by detergent treatment and flotation centrifugation. Fractional flotation analysis demonstrated that ZIKV proteins NS2B, NS3, and NS5, and ZIKV RNA were present in the detergent-resistant membranous fraction. In contrast, the ER-resident protein calnexin and a mitochondrial protein were present in the detergent-soluble fractions. The isolated RCs were functional for ZIKV RNA synthesis, as shown by quantitative PCR. To determine the components of the RCs, we conducted mass spectrometry analysis and identified numerous cellular proteins. Among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase. RFC2 is involved in ATP binding and hydrolysis and may promote cell survival. ZIKV infection increased the RFC2 protein level and induced its relocation to the cytoplasm. RNAi-mediated silencing of RFC2 reduced ZIKV replication. Together, our results provide insights into ZIKV replication and virus-cell interaction. IMPORTANCEBy isolating and characterizing the ZIKV replication complexes (RCs) from infected Vero cells, this research provides valuable insights into the viruss strategy for hijacking host intracellular membranes and proteins. The identification of ZIKV proteins (NS2B, NS3, and NS5) and RNA in detergent-resistant, lipid-rich membranous structures and the confirmation of functional RCs in RNA synthesis demonstrate successful RC purification. Mass spectrometry and proteomic analysis of the RCs identified numerous cellular proteins that may contribute to ZIKV RC formation and RNA synthesis. Notably, among them is the replication factor C subunit 2 (RFC2), an accessory protein of DNA polymerase in the nucleus. Our results demonstrate that ZIKV increases the level of RFC2 and relocates it to the cytoplasm and that RFC2 plays a role in ZIKV replication, as its depletion reduces viral yield. These results provide insights into ZIKV-cell interactions and potentially facilitate future development of novel antiviral strategies.

microbiology↗

bioRxiv2026

A horizontally transferred fungal deubiquitinase facilitates insecticides resistance in whitefly

Horizontal gene transfer (HGT) has enabled insects to acquire novel genetic material that can fuel adaptation to environmental change. However, the role of HGT in the evolution of insecticide resistance remains poorly characterised. Here, we identify BtUCH19, a fungal gene that has integrated into the genome of the global pest Bemisia tabaci and functions as a deubiquitinating enzyme (DUB). We show that compared to endogenous DUB, BtUCH19 specifically removes K63-linked ubiquitin chains from a cytochrome P450, CYP4C64, thereby stabilizing this key detoxification enzyme in vivo. Sustained CYP4C64 abundance enhances metabolic detoxification of two commonly used insecticides thiamethoxam (TMX) and clothianidin (CLO) to their low-toxicity products TMX-Urea and CLO-Urea. Consequently, BtUCH19 drives insecticide resistance through a novel "HGT-PTM-Metab" axis. Our work reveals the instrumental role of a HGT in orchestrating ubiquitin-proteasome system (UPS)-mediated protein level regulation of a key detoxification pathway. These findings offer new insights into the intricate mechanisms underlying co-evolutionary biology, and provide a new molecular target for Integrated Pest Management (IPM).

zoology↗

bioRxiv2025

Endogenous corazonin signaling modulates the post-mating switch in behavior and physiology in females of the brown planthopper and Drosophila

Mating in insects typically triggers a post-mating response (PMR) in females, characterized by reduced receptivity to re-mating and increased oviposition, which ensures numerous and viable offspring and male paternity. This PMR is induced by male seminal factors, such as sex peptide in Drosophila melanogaster, as well as intrinsic female signaling components. The latter signaling remains poorly understood in most insects, including the devastating rice pest, the brown planthopper (BPH) Nilaparvata lugens. Here, we show that the neuropeptide corazonin (CRZ) and its receptor (CrzR) are critical for the PMR in female BPHs. Peptide injection, RNAi knockdown, and CRISPR/Cas9 mutagenesis confirm that intact CRZ signaling reduces re-mating frequency and increases ovulation in mated BPH females. The CrzR is highly expressed in the female reproductive tract, and CrzR knockdown phenocopies CRZ diminishment. Importantly, female CRZ/CrzR signaling is required for male seminal factors, such as the peptide maccessin, to induce the PMR; with disrupted CrzR signaling, injection of seminal fluid or maccessin fails to reduce female receptivity. Notably, CRZ is not produced in male accessory glands (MAGs) of BPHs and thus not transferred during copulation. We furthermore demonstrate that also in D. melanogaster disrupted CRZ signaling increases female re-mating and reduces oviposition, while CRZ injection suppresses virgin receptivity and increases oviposition. Finally, we detected no CRZ in the MAG of D. melanogaster, supporting its role as an endogenous signal in the female PMR also in this species. In summary, our findings reveal a conserved role of endogenous CRZ signaling in regulating the female PMR and demonstrate that female CRZ signaling and male-derived signals cooperate to induce post-mating transitions in BPHs and D. melanogaster. CRZ is a paralog of the peptide gonadotropin-releasing hormone, known to regulate reproduction in vertebrates, including humans, suggesting evolutionary conservation of an ancient function.

genetics↗

bioRxiv2025

Dynamic Regulation of Cholesterol Metabolism Genes in Dopaminergic Neurons following Methamphetamine Treatment as Revealed by Single-Nucleus RNA Sequencing

Methamphetamine profoundly alters the function of midbrain dopaminergic neurons, yet the molecular mechanisms underlying these effects are not fully understood. Using single-nucleus RNA sequencing, we found that acute methamphetamine exposure leads to a marked up-regulation of cholesterol metabolism genes in dopaminergic neurons of the ventral tegmental area and substantia nigra--a response that was much less pronounced in astrocytes and largely absent in other cell types. Further analysis using a ribosome tagging strategy combined with RNA sequencing revealed that amphetamine, a structurally similar psychostimulant, induced similar gene expression changes, whereas methylphenidate, a structurally distinct psychostimulant, did not, highlighting drug-specific transcriptional responses. Notably, repeated methamphetamine exposure resulted in down-regulation of cholesterol metabolism genes in dopaminergic neurons. Interestingly, we also observed that, compared to neighboring cell types, dopaminergic neurons are highly enriched in genes encoding cholesterol biosynthesis enzymes, including the rate-limiting enzyme Hmgcr, and key regulators Srebf2 and Insig1, challenging the prevailing view that neurons rely mainly on astrocyte-derived cholesterol. In summary, our study highlights dynamic changes in cholesterol metabolism in dopaminergic neurons in response to amphetamines and uncovers the potential importance of cholesterol homeostasis for dopaminergic neuron function.

genomics↗

bioRxiv2025

Earlier leaf out and loss of cold tolerance for northeastern U.S. trees in response to winter warming events and early springs

O_LIWinter warming events and early springs can advance spring phenology of trees. However, these events can also decrease cold tolerance, leaving them vulnerable to subsequent refreezing. The goal of this study was to determine how spring phenology and cold tolerance of ten tree species in the northeastern U.S. responded to warming events of different timing and duration. C_LIO_LIWe exposed 300 tree seedlings of ten species to six different warming treatments. We assessed budburst, leaf out, vigor, and freezing damage on a weekly basis and measured the cold tolerance of species exposed to warming. C_LIO_LIThe timing of leaf out varied among species, and generally warming treatments advanced leaf out 5 - 41 days. Leaf out was best predicted by photoperiod for deciduous trees and thermal time for evergreen trees. Cold tolerance was reduced for most species (5 - 29 {degrees}C) when exposed to only two weeks of warming, reducing their safety margin from freezing damage. C_LIO_LIWinter warming events and early springs will drive species-specific and foliar habit responses in phenology and cold tolerance that are likely to shape competitive forest dynamics, especially at forest ecotones. C_LI

plant biology↗

bioRxiv2024

A novel male accessory gland peptide reduces female post-mating receptivity in the brown planthopper

Mating in insects commonly induces a profound change in the physiology and behavior of the female that serves to secure numerous and viable offspring and to ensure paternity for the male by reducing receptivity of the female to further mating attempts. Here, we set out to characterize the post-mating response (PMR) in a pest insect, the brown planthopper (BPH) Nilaparvata lugens and to identify a functional analog of sex peptide (SP) and/or other seminal fluid factors that contribute to the PMR in Drosophila. We find that BPHs display a distinct PMR that lasts for about 4 days and includes a change in female behavior with decreased receptivity to males and increased oviposition. Extract from male accessory glands (MAG) injected into virgin females triggers a similar PMR, lasting about 24h. Since SP does not exist in BPHs, we screened for candidate mediators by performing a transcriptional and proteomics analysis of MAG extract. We identified a novel 51 amino acid peptide present only in the MAG and not in female BPHs. This peptide, that we designate maccessin (macc), affects the female PMR. Females mated by males with macc knockdown display receptivity to wild type males in a second mating, which does not occur in controls. However, oviposition is not affected. Injection of recombinant macc reduces female receptivity, with no effect on oviposition. Thus, macc is so far the only candidate seminal fluid peptide that promotes a PMR in BPHs. Our analysis suggests that the gene encoding the macc precursor is restricted to species closely related to BPHs. Author summaryIn insects, mating often induces a long-lasting change in the female behavior and physiology, called a post-mating response (PMR). This ensures numerous and viable offspring, but also serves to secure paternity for the male by inhibiting the female receptivity to further mating attempts. Here, we demonstrate that a pest insect, the brown planthopper (BPH) Nilaparvata lugens, also displays a PMR with decreased receptivity to further mating and increased egg laying. We furthermore find that seminal fluid extracted from the male accessory gland of BPHs injected into females generates a PMR. Next, we identified a novel peptide unique to the male accessory gland (designated maccessin) and demonstrate that this peptide is responsible for the reduced receptivity in the PMR, but does not affect egg laying. The gene encoding maccessin appears unique to close relatives of N. lugens. This is similar to a Drosophila male accessory gland factor, sex peptide, which is known to induce a PMR, and occurs only in a limited number of Drosophila species.

neuroscience↗

bioRxiv2024

Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo

The most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is a G4C2 repeat expansion in intron 1 of the C9orf72 gene. This repeat expansion undergoes bidirectional transcription to produce sense and antisense repeat RNA species. Both sense and antisense-derived repeat RNAs undergo repeat-associated non-AUG translation in all reading frames to generate five distinct dipeptide repeat proteins (DPRs). Importantly, toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs. This suggests targeting both sense and antisense repeat RNA may provide the most effective therapeutic strategy. The RNA-targeting CRISPR-Cas13 systems offer a promising avenue for simultaneous targeting of multiple RNA transcripts, as they mature their own guide arrays, thus allowing targeting of more than one RNA species from a single construct. We show that CRISPR-Cas13d originating from Ruminococcus flavefaciens (CasRx) can successfully reduce C9orf72 sense and antisense repeat transcripts and DPRs to background levels in HEK cells overexpressing C9orf72 repeats. CRISPR-CasRx also markedly reduced the endogenous sense and antisense repeat RNAs and DPRs in three independent C9orf72 patient-derived iPSC-neuron lines, without detectable off-target effects. To determine whether CRISPR-CasRx is effective in vivo, we treated two distinct C9orf72 repeat mouse models using AAV delivery and observed a significant reduction in both sense and antisense repeat-containing transcripts. Taken together this work highlights the potential for RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

neuroscience↗

bioRxiv2023

Exogenous Methylglyoxal Improves Maize Yield by Alleviating Plant Diabetes and leaf senescence under Drought

Drought-induced leaf senescence is related to high sugar levels in leaves, photosynthesis inhibition, and ultimate yield loss. This physiological phenomenon in leaves bears resemblance to the symptom of diabetes in human disease. However, the underlying mechanisms of plant diabetes on carbon imbalance in maize leaf and corresponding detoxification strategy have not been well understood. In this study, we demonstrated that foliar application of exogenous methylglyoxal (MG) delayed leaf senescence and promoted photoassimilation, retrieved 14% yield loss induced by drought stress during grain filling stage. Transcriptome and metabolite analysis revealed that drought increased sugar accumulation in leaf with inhibition of sugar transporters facilitating phloem loading. This further lead to disequilibrium of glycolysis and over-accumulation of endogenous MG. Contrarily, exogenous MG significantly upregulated glycolytic flux and glyoxalase system catabolizing endogenous MG and advanced glycation end products toxicity, ultimately alleviating plant diabetes. Besides, the genes facilitating anabolism and catabolism of trehalose- 6-phosphate were promoted and suppressed by drought, respectively, whereas exogenous MG reversed the effect. Moreover, exogenous MG activated phenylpropanoid biosynthetic pathway, likely promoting cell structural integrity. Collectively, these results suggest that exogenous MG alleviates the toxic effect from drought-induced sugar accumulation and activates the defense-related pathway, thereby maintaining leaf function and yield production. HighlightExogenous methylglyoxal stimulates glycolytic flux and glyoxalase system, providing a potential insight to alleviate plant diabetes under drought condition.

physiology↗

bioRxiv2023

Rapid and accurate multi-phenotype imputation for millions of individuals

Deep phenotyping can enhance the power of genetic analysis, including genome-wide association studies (GWAS), but the occurrence of missing phenotypes compromises the potential of such resources. Although many phenotypic imputation methods have been developed, the accurate imputation of millions of individuals remains extremely challenging. In the present study, we developed a novel multi-phenotype imputation method based on mixed fast random forest (PIXANT) by leveraging efficient machine learning (ML)-based algorithms. We demonstrate that PIXANT runtime is faster and computer memory usage is less than that of other state-of-the-art methods when applied to the UK Biobank (UKB) data, suggesting that PIXANT is scalable to cohorts with millions of individuals. Our simulations with hundreds of individuals showed that PIXANT accuracy was superior to or comparable to the accuracy of the most advanced methods available. PIXANT was used to impute 425 phenotypes for the UKB data of 277,301 unrelated White British citizens. When GWAS was subsequently performed on the imputed phenotypes, 18.4% more GWAS loci were identified than before imputation (8,710 vs 7,355). The increased statistical power of GWAS identified novel positional candidate genes affecting heart rate, such as RNF220, SCN10A, and RGS6, suggesting that the use of imputed phenotype data from a large cohort may lead to the discovery of novel genes for complex traits.

bioinformatics↗

bioRxiv2023

Comparison of shear stress patterns by the established and advanced reconstruction method incorporating side branches to predict plaque progression

BackgroundComplete vessel reconstruction (CVR) with incorporation of side branches is essential for accurate evaluation of wall shear stress (WSS) distribution. However, CVR is time consuming and blood flow simulation is computationally expensive, while there is no evidence that WSS computed by CVR, enables better prediction of disease progression compared to WSS derived from the conventional single vessel reconstruction (SVR). We aim to compare the WSS in models reconstructed using the CVR and SVR methods and examine its ability to predict disease progression. MethodsPatients who had baseline and 13-months follow-up intravascular ultrasound (IVUS) imaging (n=19 vessels), and with neoatherosclerotic lesions (n=13 vessels) on optical coherence tomography (OCT) were included in the present analysis. All the studied vessels had at least one side branch with diameter >1mm. 3-dimensional (3D) CVR and SVR were performed and time averaged (TAWSS) and multidirectional WSS were computed using pulsatile blood flow simulation and the performance of both methods in predicting disease progression in IVUS and OCT models were assessed. ResultsThe incorporation of side branches in 3D geometry resulted in lower TAWSS in the IVUS (0.821 vs 1.698Pa, p<0.001) and OCT-based reconstructions (0.682 vs 1.325Pa, p<0.001) and influenced the multidirectional WSS distribution. In native segments, WSS metrics estimated by the CVR enabled better prediction of the lumen and plaque area and burden at follow-up than SVR and disease progression defined as decrease in lumen area and increase in plaque burden (AUC CVR 0.712 vs SVR 0.554). In stented segments, multidirectional WSS was associated with neointima area in both CVR and SVR methods, but TAWSS was only a predictor of neointima area in the CVR method. ConclusionsThe incorporation of side branches in vessel reconstruction influences WSS distribution and enables more accurate prediction of disease progression in native and stented segments than SVR modelling. HighlightsO_LIComplete vessel reconstruction (CVR) with incorporation of vessel side branches has been proposed for accurate evaluation of wall shear stress (WSS) distribution compared to the traditional single vessel reconstruction (SVR) method; however, there are no studies comparing the performance of the WSS metrics derived by these methods in predicting atherosclerotic evolution. C_LIO_LIIn vessels with large side branches, the incorporation of the side branches in the vessel geometry reconstructed from angiographic and intravascular imaging data resulted in lower time averaged wall shear stress (TAWSS) and influenced the multidirectional WSS estimations compared to the models reconstructed without the side branches. C_LIO_LIThe WSS metrics estimated in the CVR models enabled better prediction of atherosclerotic disease progression at 13-months follow-up on IVUS than the WSS derived by the SVR. C_LIO_LIIn stented vessels, all the WSS metrics in the CVR and the multidirectional WSS in SVR were associated with neointima tissue development; however, both approaches showed limited efficacy in predicting neointima proliferation. C_LI

bioengineering↗

bioRxiv2022

Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration

Mitochondrial dysfunction and axon loss are hallmarks of neurologic diseases. Gasdermin (GSDM) proteins are executioner pore-forming molecules that mediate cell death, yet their roles in the central nervous system (CNS) are not well understood. Here, we find that one GSDM family member, GSDME is expressed by both mouse and human neurons. GSDME plays a role in mitochondrial damage and axon loss. Mitochondrial neurotoxins induced caspase-dependent GSDME cleavage and rapid localization to mitochondria in axons, where GSDME promoted mitochondrial depolarization, trafficking defects, and neurite retraction. The frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS)-associated proteins TDP-43 and PR-50 induced GSDME-mediated damage to mitochondria and neurite loss. GSDME deficiency prolonged survival, ameliorated motor dysfunction, and rescued motor neuron loss in the SOD1G93A mouse model of ALS. GSDME knockdown also protected against neurite loss in ALS patient iPSC-derived motor neurons. Thus, we identify GSDME as an executioner of neuronal mitochondrial dysfunction that contributes to neurodegeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/513927v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@113c13dorg.highwire.dtl.DTLVardef@1f3c5caorg.highwire.dtl.DTLVardef@13e75adorg.highwire.dtl.DTLVardef@19fdc93_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGSDME is expressed by neurons and activated by mitochondrial neurotoxins C_LIO_LIActivated GSDME drives axonal mitochondrial damage and neurite loss prior to cell death C_LIO_LIALS/FTD associated TDP-43 and PR-50 induces GSDME-driven neurite loss in mouse and human iPSC-derived neurons. C_LIO_LISOD1G93A mice show ameliorated disease progression and motor neuron loss in absence of GSDME C_LI

neuroscience↗

bioRxiv2022

Influence of amino acids and glucosinolates in three Brassicaceae vegetable plants on preference and performance of the green peach aphid: Myzus persicae

The green peach aphid, Myzus persicae (Sulzer) is a generalist pest of various host plants, whose feeding preference and growth performance mainly depends on the quantity and quality of nutrients and defensive metabolites in host plants. Here, we studied the preference and performance of M. persicae on three major Brassicaceae vegetables in China and measured nutrient (amino acids) and defensive (glucosinolates) metabolites in these plants. We found that M. persicae preferred and performed better on Chinese cabbage than cabbage and radish, which may be due to the relatively higher concentration of amino acids and lower levels of glucosinolates in their leaves. The glucosinolates level in cabbage leaves was ten times higher than the other two plants, while the amino acid concentration in radish was only half of the cabbage or Chinese cabbage. The higher concentration of glucosinolates in cabbage and lower levels of amino acids in radish may account for the poorer preference and growth of M. persicae on these two plants. These results suggest that both amino acids and glucosinolates in plants are important determinants of the preference and performance of M. persicae, which provide new knowledge for the cultivation and breeding of Brassicaceae vegetables.

ecology↗

bioRxiv2022

Inflammasome mediated neuronal-microglial crosstalk: a therapeutic substrate in C9orf72-FTD/ALS.

Intronic G4C2 hexanucleotide repeat expansions (HRE) of C9orf72 are the most common cause of familial variants of frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). G4C2 HREs in C9orf72 undergo non-canonical repeat-associated translation, producing dipeptide repeat (DPR) proteins, with various deleterious impacts on cellular homeostasis. While five different DPRs are produced, poly(glycine-arginine) (GR) is amongst the most toxic and is the only DPR to accumulate in the associated clinically relevant anatomical locations of the brain. Previous work has demonstrated the profound effects of a poly(GR) model of C9orf72 FTD/ALS, including motor impairment, memory deficits, neurodegeneration, and neuroinflammation. Neuroinflammation is hypothesized to be a driving factor in the disease course; microglia activation is present prior to symptom onset and persists throughout the disease. Here, using an established mouse model of C9orf72 FTD/ALS we investigate the contributions of the nod-like receptor pyrin-containing 3 (NLRP3) inflammasome in the pathogenesis of FTD/ALS. We find that inflammasome-mediated neuroinflammation is increased with microglial activation, cleavage of caspase-1, production of IL-1{beta} and upregulation of Cxcl10 in the brain of C9orf72 FTD/ALS mice. Excitingly, we find that genetic ablation of Nlrp3 significantly improved survival, protected behavioral deficits and prevented neurodegeneration suggesting a novel mechanism involving HRE-mediated induction of innate immunity. The findings provide experimental evidence of the integral role of HRE in inflammasome-mediated innate immunity in the C9orf72 variant of FTD/ALS pathogenesis and suggest the NLRP3 inflammasome as a therapeutic target.

neuroscience↗

bioRxiv2022

Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease

Hexanucleotide G4C2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model. SignificanceImmunotherapy has been proposed for neurodegenerative disorders including Alzheimers or Parkinsons diseases. Recent reports using antibodies against poly-GA or active immunization suggested similar immunotherapy in ALS/FTD caused by repeat expansion in the C9ORF72 gene (1, 2). Here, we systematically characterized human antibodies against multiple DPR species and tested the biological effects of antibodies targeting poly-GA in different cellular and mouse models. Target engagement was shown in three independent cellular models. Anti-GA antibodies reduced the number of intracellular poly-GA aggregates in human T98G cells but not in cultured human neurons. Whereas chronic anti-GA treatment in BAC C9ORF72450 mice did not impact poly-GA levels and modestly improved one behavioral phenotype, poly-GA levels detected by immunoassays were increased and disease progression was unaltered in AAV-(G4C2)149 mice.

neuroscience↗

bioRxiv2020

YTHDF2 is essential for spermatogenesis and fertility through mediating a wave of transcript transition during spermatogonial differentiation

The dynamic and reversible regulation roles of m6A modification, and the characterization of m6A readers have provided new insights into spermatogenesis at post-transcriptional level. YTHDF2 has been reported to recognize and mediate the m6A-containing transcripts decay during the mouse oocyte mature, embryonic stem cell differentiation, neural development, and zebrafish maternal-to-zygotic transition. However, the roles of YTHDF2 in mammalian spermatogenesis are uncertain. Here, we generated germ cell-specific Ythdf2 mutants (Ythdf2-vKO) at a C57BL/6J background, and demonstrated that YTHDF2 was essential for mouse spermatogenesis and fertility. Ythdf2-vKO provided oligoasthenoteratozoospermia (OAT) phenotype with increased apoptosis in germ cells. High-throughput RNA-seq of the testis tissue showed the failure of the degradation of a wave of YTHDF2 target mRNA. Interestingly, RNA-seq analysis combined with our previous single-cell transcriptomics data of mouse spermatogenesis pointed out the failure of a wave of transcript transition during the spermatogenesis of Ythdf2-vKO, which was confirmed by gene expression analysis of diplotene spermatocytes and round spermatids obtained through fluorescence-activated cell sorting using qPCR. Our study demonstrates the fundamental role of YTHDF2 during mouse spermatogenesis and provides a potential candidate for the diagnosis of male infertility with OAT syndrome. Author summaryMale infertility is becoming a worldwide health problem. Male gamete is generated through spermatogenesis, which is a complicated developmental process with dynamic transcriptome changes. RNA m6A modification has been reported as the most prominently internal mRNA modification, which control the tune of gene expression through mRNA splicing, export, translation and decay. RNA m6A modification is catalyzed by "writers", and could be removed by "erasers". The m6A modification enzymes are reported to play important roles during spermatogenesis. Given that the biological function of m6A modification are mediated through its "readers", its readers might be proposed to be involved in the regulation of spermatogenesis. YTHDF2, as a reader of m6A modification, has been reported to mediate the m6A-containing transcripts decay. To explore the roles of YTHDF2 in spermatogenesis, we used germ cell-specific mutation strategy to knock out the mouse Ythdf2. The mutants provided oligoasthenoteratozoospermia phenotype. Our study demonstrate that YTHDF2 is essential for mouse spermatogenesis. YTHDF2 could be a potential candidate for the diagnosis of oligoasthenoteratozoospermia syndrome.

genetics↗

bioRxiv2018

Modeling circRNAs expression pattern with integrated sequence and epigenetic features identifies H3K79me2 as regulators for circRNAs expression

Circular RNAs (circRNAs) are an abundant class of noncoding RNAs with widespread, cell/tissue specific pattern. Because of their involvement in the pathogenesis of multiple disease, they are receiving increasing attention. Previous work suggested that epigenetic features might be related to circRNA expression. However, current algorithms for circRNAs prediction neglect these features, leading to constant results across different cells.\n\nHere we built a machine learning framework named CIRCScan, to predict expression status and expression levels of circRNAs in various cell lines based on sequence and epigenetic features. Both expression status and expression levels can be accurately predicted by different groups of features. For expression status, the top features were similar in different cells. However, the top features for predicting expression levels were different in different cells. Noteworthy, the importance of H3K79me2 ranked high in predicting both circRNAs expression status and levels across different cells, indicating its important role in regulating circRNAs expression. Further validation experiment in K562 confirmed that knock down of H3K79me2 did result in reduction of circRNA production.\n\nOur study offers new insights into the regulation of circRNAs by incorporating epigenetic features in prediction models in different cellular contexts.

bioinformatics↗

bioRxiv2017

Vegetally Localized Vrtn Functions As A Novel Repressor To Modulate bmp2b Transcription During Dorsoventral Patterning In Zebrafish

The vegetal pole cytoplasm represents a critical source of maternal signals for patterning the primary dorsoventral axis of the early embryo. Vegetally localized dorsal determinants are essential for the formation of the Spemann organizer, which expresses both bone morphogenetic proteins and their antagonists. The extracellular regulation of BMP signalling activity has been well characterized, however, transcriptional regulation of bmp genes along the dorsoventral axis remains largely unknown. Here, we report a novel mode of maternal regulation of BMP signalling in the lateral and ventral regions by analyzing the unexpected dorsalizing effect following vegetal yolk ablation experiment. We identified Vrtn as a novel vegetally localized maternal factor displaying dorsalizing activity. It functions as a novel transcriptional repressor to regulate bmp2b expression in the marginal region. Mechanistically, Vrtn binds bmp2b upstream sequence and inhibits its transcription independently of maternal Wnt/{beta}-catenin signalling. By creating vrtn loss-of-function mutation and analyzing maternal-zygotic mutant embryos, we further showed that Vrtn is required for the formation of dorsoventral axis. Our work thus unveils a novel maternal mechanism regulating BMP gradient during dorsoventral specification.

developmental biology↗