Biology subjects

Zhuang, X.

Publications and source records attributed to Zhuang, X..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Positive and relaxed selective pressures have both strongly influenced the evolution of cryonotothenioid fishes during their radiation in the freezing Southern Ocean

Evolution in the chronic cold of the Southern Ocean has had a profound influence on the physiology of cryonotothenioid fishes. However, the suite of genetic changes underlying the physiological gains and losses in these fishes is still poorly surveyed. Using molecular evolution techniques, this study aimed to identify which functional classes of genes changed during the cryonotothenioid radiation in a polar ocean. The influences of both positive and relaxed selective pressures were isolated following two major physiological transitions: the onset of freezing temperatures, and the loss of hemoproteins. Looking at the influence of cold temperatures, positive selective pressure was most prominently found to act on biosynthetic processes (the machinery of transcription and translation) as well as on protein polymerization, cell adhesion, and cell cycle progression, suggesting these are key challenges to life in freezing waters. Relaxation of selective pressure produced a more complex pattern of change, affecting several homeostatic processes, suggesting their attenuation in cold-stable and oxygen-rich waters, but also seemingly acting as a route to key genetic change behind the morphological and ecological diversification seen in the clade. Finally, while chronic cold-water temperatures appear to have instigated clear genetic change, the loss of hemoproteins led to little observable change relative to their red-blooded relatives. Combined, the influence of positive and relaxed selection show that long-term exposure to cold has led to profound changes in cryonotothenioid genomes, which may make it challenging for them to respond to unpredictable heat waves and to adapt to a rapidly changing climate.

evolutionary biology↗

bioRxiv2022

PERSISTENT RELIEF OF MOTOR SYMPTOMS IN A PARKINSONIAN MOUSE MODEL AFTER INDUCTION OF LTD AT CORTICAL INPUTS TO INDIRECT PATHWAY STRIATAL NEURONS

In Parkinsons disease (PD) patients, dopamine replacement therapy requires days to reach maximal effects, and the return of symptoms without treatment is similarly delayed. We previously postulated that these phenomena are mediated by plasticity of coritcostriatal synapses. As dopamine depletion is expected to promote aberrant potentiation of the cortical inputs onto indirect pathway neurons, we reasoned that induction of LTD here could reduce motor deficits in a PD model. Optogenetic cortical stimulation combined with a D2 receptor agonist, quinpirole, induces robust optical LTD (oLTD) in brain slices from 6-OHDA lesioned mice. When lesioned mice were subjected to corticostriatal oLTD treatment over 5 days, motor performance was improved for >3 weeks. Consistent with LTD induction, oLTD-treated mice had reduced VGLUT1 expression in striatum and greater excitability of D2 neurons. These findings suggest that reversing aberrant corticostriatal synaptic plasticity in the indirect pathway may lead to persistent relief of PD motor symptoms.

neuroscience↗

bioRxiv2022

Spatially resolved single-cell deconvolution of bulk RNA-seq using Bulk2Space

Uncovering the tissue molecular architecture at single-cell resolution could help better understand organisms biological and pathological processes. However, bulk RNA-seq can only measure gene expression in cell mixtures, without revealing the transcriptional heterogeneity and spatial patterns of single cells. Herein, we introduce Bulk2Space (https://github.com/ZJUFanLab/bulk2space), a deep learning framework-based spatial deconvolution algorithm that can simultaneously disclose the spatial and cellular heterogeneity of bulk RNA-seq data using existing single-cell and spatial transcriptomics references. The use of bulk transcriptomics to validate Bulk2Space unveils, in particular, the spatial variance of immune cells in different tumor regions, the molecular and spatial heterogeneity of tissues during inflammation-induced tumorigenesis, and spatial patterns of novel genes in different cell types. Moreover, Bulk2Space is utilized to perform spatial deconvolution analysis on bulk transcriptome data from two different mouse brain regions derived from our in-house developed sequencing approach termed Spatial-seq. We have not only reconstructed the hierarchical structure of the mouse isocortex but also further annotated cell types that were not identified by original methods in the mouse hypothalamus.

bioinformatics↗

bioRxiv2021

Trapping of Nicotinic Acetylcholine Receptor Ligands Assayed by in vitro Cellular Studies and in vivo PET Imaging

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membranepermeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKa and high-affinity for 4{beta}2-type nicotinic receptors (4{beta}2Rs) are trapped in intracellular acidic vesicles containing 4{beta}2Rs in vitro. Nicotine, with lower pKa and 4{beta}2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18F-labelled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKa and affinity and [18F]Nifene with nicotine-like pKa and affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in 4{beta}2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consistent with its binding to 4{beta}2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in {beta}2 subunit knockout mice or by acute nicotine injections demonstrating binding to sites on {beta}2-containing receptors. Chloroquine, which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributions in vivo consistent with only [18F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in 4{beta}2R-containing GSats, which results in very slow release long after nicotine is gone after smoking. SignificanceMechanisms of nicotine addiction remain poorly understood. An earlier study using in vitro methods found that the anti-smoking nicotinic ligand, varenicline (Chantix) was trapped in 4{beta}2R-containing acidic vesicles. Using a fluorescent labeled high-affinity nicotinic ligand, this study provided evidence that these intracellular acidic vesicles were 4{beta}2R-containing Golgi satellites. In vivo PET imaging with F-18 labeled nicotinic ligands provided additional evidence that differences in PET ligand trapping in acidic vesicles were the cause of differences in PET ligand kinetics and subcellular distributions. These findings combining in vitro and in vivo imaging revealed new mechanistic insights into the kinetics of weak base PET imaging ligands and the subcellular mechanisms underlying nicotine addiction.

neuroscience↗

bioRxiv2021

Conservation and divergence in cortical cellular organization between human and mouse revealed by single-cell transcriptome imaging

The human cerebral cortex has tremendous cellular diversity and complex cellular organization that are essential for brain function. How different types of cells are organized and interact with each other in the human cortex, and how cellular organizations and interaction patterns vary across species are, however, unclear. Here, we performed spatially resolved single-cell expression profiling of 4,000 genes in human middle and superior temporal gyrus using multiplexed error-robust fluorescence in situ hybridization (MERFISH). We identified >100 neuronal and non-neuronal cell populations with distinct transcriptional signatures, generated a molecularly defined and spatially resolved cell atlas of these brain regions, and analyzed cell-cell interactions in a cell-type-specific manner. Comparison with the mouse cortex showed conservation in the laminar organization of cells and substantial divergence in cell-cell interactions between human and mouse. Notably, our data revealed a drastic increase in interactions between neurons and non-neuronal cells in the human cortex, uncovered human-specific cell-cell interaction patterns, and identified potential ligand-receptor basis of microglia-neuron interactions.

neuroscience↗

bioRxiv2021

Chronic alcohol drinking persistently suppresses thalamostriatal excitation of cholinergic neurons to impair cognitive flexibility

Exposure to addictive substances impairs flexible decision-making. Cognitive flexibility is mediated by striatal cholinergic interneurons (CINs). However, how chronic alcohol drinking alters cognitive flexibility through CINs remains unclear. Here, we report that chronic alcohol consumption and withdrawal impaired reversal of instrumental learning. Chronic alcohol consumption and withdrawal also caused a long-lasting (21 d) reduction of excitatory thalamic inputs onto CINs and reduced pause response of CINs in the dorsomedial striatum (DMS). CINs are known to inhibit glutamatergic transmission in dopamine D1 receptor-expressing medium spiny neurons (D1-MSNs) but facilitate this transmission in D2-MSNs, which may contribute to flexible behavior. We discovered that chronic alcohol drinking impaired CIN-mediated inhibition in D1-MSNs and facilitation in D2-MSNs. Importantly, in vivo optogenetic induction of long-term potentiation of thalamostriatal transmission in DMS CINs rescued alcohol-induced reversal learning deficits. These results demonstrate that chronic alcohol drinking reduces thalamic excitation of DMS CINs, compromising their regulation of glutamatergic transmission in MSNs, which may contribute to alcohol-induced impairment of cognitive flexibility. These findings provide a neural mechanism underlying inflexible drinking in alcohol use disorder.

neuroscience↗

bioRxiv2021

Towards Understanding Comprehensive Morphometric Changes and Its Correlation with Cognition and Exposure to Fighting in Active Professional Boxers

Professional athletes exposed to repetitive head impacts are at increased risk for developing a progressive neurological syndrome known as traumatic encephalopathy syndrome and neuropathology seen on autopsy called chronic traumatic encephalopathy (CTE). The early identification of individuals at increased risk for CTE is important and the search for biomarkers is underway. In this study, we utilized data from a large cohort study to compare differences in regional brain volumes, cortical thickness, voxel-based morphometric (VBM)-derived measures, and graph-theoretical measures derived from large-scale topographical maps in active professional boxers. We compared the above morphometric measures between active professional boxers with low cognitive scores (impaired boxers) and active professional boxers with intact cognitive scores (nonimpaired boxers). The cognitive scores were evaluated through neuropsychological evaluation. As an exploratory analysis, we also examined the power of various machine-learning algorithms to identify impaired and nonimpaired boxers using both group-level regression-driven analysis and previously identified hypothesis-driven cortical thickness and volumetric measures. We found significant group-level differences between impaired and nonimpaired boxers in cortical thickness in a single brain region (right precuneus), differences in VBM-derived gray matter density encompassing the caudate, putamen, and thalamus; and white matter density encompassing the right paracentral lobule, but no differences in any graph-theoretical network properties. Additionally, we found that a priori hypothesis-driven T1-derived cortical thickness and volumetric analysis performed better than traditional regression-based analysis. Overall, this study suggests that neuroanatomical differences exist between impaired and nonimpaired active professional boxers, and that hypothesis-driven techniques are likely necessary to become reliable biomarkers.

neuroscience↗

bioRxiv2021

Diet modifies allele-specific phenotypes in Drosophila carrying epilepsy-associated PNPO variants

Pyridox(am)ine 5-phosphate oxidase (PNPO) catalyzes the rate-limiting step in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B6 required for the synthesis of neurotransmitters GABA and monoamines. Pathogenic variants in PNPO have been repeatedly identified in patients with neonatal epileptic encephalopathy and early-onset epilepsy. These patients often exhibit different types of seizures and variable comorbidities, including developmental impairment and intellectual disability. It is unclear how seizure types and associated comorbidities are linked to specific PNPO alleles and to what degree diet can modify their expression. Furthermore, the molecular characteristics of PNPO variants have not been examined in model systems. Using CRISPR/Cas9, we generated four knock-in Drosophila alleles, hWT, hR116Q, hD33V, and hR95H, in which the endogenous Drosophila PNPO (sugarlethal) was replaced by wild-type human PNPO cDNA and epilepsy-associated variants corresponding to R116Q, D33V, and R95H, respectively. We examined these knock-in flies at the molecular, circuitry, and behavioral levels. Collectively, we found a wide range of phenotypes in an allele- and diet-dependent manner. Specifically, the D33V mutation reduces the mRNA level, R95H reduces the protein stability, and R116Q alters the protein localization of PNPO in the brain. D33V and R95H mutations lead to partial and complete lethality during development, respectively and R116Q and D33V mutations shorten lifespan. At the behavioral level, hD33V/hR95H trans-heterozygous flies are hypoactive on all tested diets whereas hR116Q flies show diet-dependent locomotor activities. At the circuitry level, hD33V homozygotes show rhythmic burst firing and hD33V/hR95H trans-heterozygotes exhibit spontaneous seizure discharges. In hR95H homozygotes rescued with PLP supplementation, we uncovered that PLP deficiency abolishes development and causes extreme seizures in adults. Lastly, genetic and electrophysiological analyses demonstrate that hWT/hR95H heterozygous flies are prone to seizures due to a dominant-negative effect of hR95H on hWT, highlighting the possibility that human R95H carriers may also be susceptible to epilepsy. Together, this study demonstrates that human PNPO variants interact with diet to contribute to phenotypic variations; and that the knock-in Drosophila model offers a powerful approach to systematically examine clinical manifestations and the underlying mechanisms of human PNPO deficiency.

neuroscience↗

bioRxiv2021

Decoding molecular and cellular heterogeneity of nucleus accumbens with high-throughput scRNA-seq and MERFISH

The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. Here, we generate a cell census of the mouse NAc using high-throughput single cell RNA sequencing and multiplexed error-robust FISH, revealing a high level of cell heterogeneity in this brain region. We show that the transcriptional and spatial diversity of neuron subtypes underlie NAcs anatomic and functional heterogeneity, and possibly contribute to the pathogenesis of different neurological disorders. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the NAc structure and function, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system.

neuroscience↗

bioRxiv2021

Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/450133v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@10f7bf1org.highwire.dtl.DTLVardef@192214dorg.highwire.dtl.DTLVardef@c84916org.highwire.dtl.DTLVardef@1366287_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefBy detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain. Highlights O_LISingle molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics C_LIO_LIThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replication C_LIO_LIGenomic RNA is stable and persistent during the initial stages of infection C_LIO_LIB.1.1.7 variant replicates more slowly than the Victoria strain C_LI

microbiology↗

bioRxiv2021

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates metabolism and development via translation

Ribosomal RNAs (rRNAs) have long been known to carry modifications, including numerous sites of 2O-methylation and pseudouridylation, as well as N6-methyladenosine (m6A), and N6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry m6A, and while ZCCHC4 has been identified as the methyltransferase responsible for the 28S rRNA m6A site, the methyltransferase responsible for the 18S rRNA m6A site has remained uncharacterized until recently. Here, we show that the METTL5-TRMT112 complex is the methyltransferase responsible for installing m6A at position 1832 of human 18S rRNA. TRMT112 is required for the metabolic stability of METTL5, and human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. Loss of METTL5 in human cancer lines alters the translation of transcripts associated with mitochondrial biogenesis and function. Mettl5 knockout mice display reduced body size and evidence of metabolic defects. This m6A site is located on the 3 end of 18S rRNA, which may become surface-exposed under some circumstances and thus may play a regulatory role in translation of specific transcripts. While recent work has focused heavily on m6A modifications in mRNA and its roles in mRNA processing and translation, deorphanizing putative methyltransferase enzymes is revealing previously unappreciated regulatory roles for m6A in noncoding RNAs.

biochemistry↗

bioRxiv2021

Circadian regulation of SARS-CoV-2 infection in lung epithelial cells

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via Spike glycoprotein binding angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organisms response to its environment and can regulate host susceptibility to virus infection. We demonstrate a circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with a synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced a wide spectrum of interferon stimulated genes in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to dampen SARS-CoV-2 infection. Our study suggests new approaches to understand and improve therapeutic targeting of SARS-CoV-2.

microbiology↗

bioRxiv2020

Proteomic and functional analyses of the periodic membrane skeleton in neurons

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of candidate MPS-interacting proteins that span diverse functional categories. We validated representative proteins in several of these categories, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, demonstrating periodic distributions of [~]20 proteins in neurons using super-resolution imaging. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide new insights into the interactome of the MPS, and suggest new functions of the MPS in neurons.

neuroscience↗

bioRxiv2020

Spatial organization of the transcriptome in individual neurons

Neurons are highly polarized cells with complex neurite morphology. Spatial organization and local translation of RNAs in dendrites and axons play an important role in many neuronal functions. Here we performed super-resolution spatial profiling of RNAs inside individual neurons at the genome scale using multiplexed error-robust fluorescence in situ hybridization (MERFISH), and mapped the spatial organization of up to [~]4,200 RNA species (genes) across multiple length scales, ranging from sub-micrometer to millimeters. Our data generated a quantitative intra-neuronal atlas of RNAs with distinct transcriptome compositions in somata, dendrites, and axons, and revealed diverse sub-dendritic distribution patterns of RNAs. Moreover, our spatial analysis identified distinct groups of genes exhibiting specific spatial clustering of transcripts at the sub-micrometer scale that were dependent on protein synthesis and differentially dependent on synaptic activity. Overall, these data provide a rich resource for characterizing the subcellular organization of the transcriptome in neurons with high spatial resolution.

cell biology↗

bioRxiv2020

Cocaine causes rapid remodeling of dopaminergic axons, synapses, and mitochondria

Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change, for example, with drug exposure, remains poorly understood. We combine recent advances in cell type specific labeling for electron microscopy with large volume three-dimensional serial electron microscopy - connectomics- to detail DA connections in the Nucleus Accumbens (NAc) across multiple animals and after exposure to cocaine. We find that DA axonal varicosities are of four general types: 38% are empty, 25% contain few small ([~]50 nm diameter) vesicles, 19% contain few large ([~]133 nm diameter) vesicles, and 18% have mixed small and large vesicles, suggesting that DA axons may use multiple types of neurotransmitters. Individual DA axons were significantly more likely to contain multiple varicosities of the same type relative to chance, suggesting a new method of DA axon classification. Across all categories, we find only rare examples (<2%, 6/410) of varicosities making specific synapses with any neighboring neuron with the few examples being made exclusively on the shafts and soma of resident NAc neurons. Instead, we find much more frequently (15%) that DA varicosities form spinule-like structures: physical membrane interdigitations with nearby dendrites or excitatory and inhibitory axons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls but with similar densities of varicosities and spinules. Additionally, cocaine exposure results in the formation of blind-ended "bulbs" in DA axons, filled with mitochondria, and reminiscent of axonal retraction in the developing and damaged brain. Every bulb was surrounded by elaborated glia further suggestive of active remodeling. Finally, mitochondrial lengths increased by [~]2.2 times relative to control throughout DA axons and NAc spiny dendrites after cocaine exposure but not in DA soma or DA dendrites. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large scale axonal rearrangements with correlated changes in mitochondria.

neuroscience↗

bioRxiv2020

Deep learning and alignment of spatially-resolved whole transcriptomes of single cells in the mouse brain with Tangram

Charting a biological atlas of an organ, such as the brain, requires us to spatially-resolve whole transcriptomes of single cells, and to relate such cellular features to the histological and anatomical scales. Single-cell and single-nucleus RNA-Seq (sc/snRNA-seq) can map cells comprehensively5,6, but relating those to their histological and anatomical positions in the context of an organs common coordinate framework remains a major challenge and barrier to the construction of a cell atlas7-10. Conversely, Spatial Transcriptomics allows for in-situ measurements11-13 at the histological level, but at lower spatial resolution and with limited sensitivity. Targeted in situ technologies1-3 solve both issues, but are limited in gene throughput which impedes profiling of the entire transcriptome. Finally, as samples are collected for profiling, their registration to anatomical atlases often require human supervision, which is a major obstacle to build pipelines at scale. Here, we demonstrate spatial mapping of cells, histology, and anatomy in the somatomotor area and the visual area of the healthy adult mouse brain. We devise Tangram, a method that aligns snRNA-seq data to various forms of spatial data collected from the same brain region, including MERFISH1, STARmap2, smFISH3, and Spatial Transcriptomics4 (Visium), as well as histological images and public atlases. Tangram can map any type of sc/snRNA-seq data, including multi-modal data such as SHARE-seq data5, which we used to reveal spatial patterns of chromatin accessibility. We equipped Tangram with a deep learning computer vision pipeline, which allows for automatic identification of anatomical annotations on histological images of mouse brain. By doing so, Tangram reconstructs a genome-wide, anatomically-integrated, spatial map of the visual and somatomotor area with [~]30,000 genes at single-cell resolution, revealing spatial gene expression and chromatin accessibility patterning beyond current limitation of in-situ technologies.

genomics↗

bioRxiv2020

Cyclin G2 inhibits oral squamous cell carcinoma growth and metastasis by binding to insulin-like growth factor binding protein 3 and regulating the FAK-SRC-STAT signaling pathway

The cell cycle protein cyclin G2 is considered a tumor suppressor. However, its regulatory effects and potential mechanisms in oral cancers are not well understood. This study aimed to investigate the effect of cyclin G2 on oral squamous cell carcinoma (OSCC). The data from 80 patients with OSCC were utilized to predict the abnormal expression of cyclin G2. The proliferation and metastasis were determined by a cell counting Kit-8 assay, flow cytometry, a wound-healing assay and a cell invasion assay. The expression of key proteins and genes associated with the cyclin G2 signaling pathways was determined by western blotting and real-time PCR, respectively. The orthotopic nude mice model was established by a mouth injection of SCC9 cells overexpressing cyclin G2. We showed that the low level of cyclin G2 in OSCC, which is negatively correlated with clinical staging, was a negative prognostic factor for the disease. We also found that cyclin G2 inhibited the proliferation, metastasis and blocked the cell cycle at G1/S of OSCC cells, suggesting that cyclin G2 has an inhibitory effect in OSCC. Mechanistically, cyclin G2 inhibited the growth and metastasis of OSCC by binding to insulin-like growth factor binding protein 3 (IGFBP3) and regulating the focal adhesion kinase (FAK) -SRC-STAT signal transduction pathway. Cyclin G2 competed with integrin to bind to IGFBP3; the binding between integrin and IGFBP3 was reduced after cyclin G2 overexpression, thereby inhibiting the phosphorylation of FAK and SRC. These results showed that cyclin G2 inhibited the progression of OSCC by interacting with IGFBP3 and that it may be a new target for OSCC treatment.

cancer biology↗

bioRxiv2020

Molecular, spatial and projection diversity of neurons in primary motor cortex revealed by in situ single-cell transcriptomics

A mammalian brain is comprised of numerous cell types organized in an intricate manner to form functional neural circuits. Single-cell RNA sequencing provides a powerful approach to identify cell types based on their gene expression profiles and has revealed many distinct cell populations in the brain1-3. Single-cell epigenomic profiling4,5 further provides information on gene-regulatory signatures of different cell types. Understanding how different cell types contribute to brain function, however, requires knowledge of their spatial organization and connectivity, which is not preserved in sequencing-based methods that involve cell dissociation3,6. Here, we used an in situ single-cell transcriptome-imaging method, multiplexed error-robust fluorescence in situ hybridization (MERFISH)7, to generate a molecularly defined and spatially resolved cell atlas of the mouse primary motor cortex (MOp). We profiled [~]300,000 cells in the MOp, identified 95 neuronal and non-neuronal cell clusters, and revealed a complex spatial map in which not only excitatory neuronal clusters but also most inhibitory neuronal clusters adopted layered organizations. Notably, intratelencephalic (IT) cells, the largest branch of neurons in the MOp, formed a continuous spectrum of cells with gradual changes in both gene expression profiles and cortical depth positions in a highly correlated manner. Furthermore, we integrated MERFISH with retrograde tracing to probe the projection targets for different MOp neuronal cell types and found that projections of MOp neurons to other cortical regions formed a many-to-many network with each target region receiving input preferentially from a different composition of IT clusters. Overall, our results provide a high-resolution spatial and projection map of molecularly defined cell types in the MOp. We anticipate that the imaging platform described here can be broadly applied to create high-resolution cell atlases of a wide range of systems.

neuroscience↗