Cryptococcus neoformans, a fungal pathogen causing fatal meningitis mainly in immunocompromised individuals. It has been shown that ZNFs play important roles in the morphological development, differentiation, and virulence of C. neoformans. In this review, we, first, briefly introduce the ZNFs and their classification. Then, we explain the identification and classification of the ZNFs in C. neoformans. Next, we focus on the biological role of the ZNFs functionally characterized so far in the sexual reproduction, virulence factor production, ion homeostasis, pathogenesis, and stress resistance in C. neoformans. We also discuss the perspectives on future function studies of ZNFs in C. neoformans.Zinc is one of the essential trace elements in eukaryotes and it is a critical structural component of a large number of proteins. Zinc finger proteins (ZNFs) are zinc-finger domain-containing proteins stabilized by bound zinc ions and they form the most abundant proteins, serving extraordinarily diverse biological functions. In recent years, many ZNFs have been identified and characterized in the human fungal pathogen  Cryptococcus neoformans is a basidiomycetous yeast pathogen that causes fungal meningitis mainly in immunocompromised individuals . B,9. BC. n. gattii ,11,12,13 species . Others tability .C. neoformans is very complicated and still need to be further explored.Up to now, many signaling pathways or factors involved in sexual reproduction and fungal virulence have been characterized and extensively studied ,18,19,20Saccharomyces cerevisiae, where there have been more than 50 ZNFs identified in this organism based on the analysis of genome sequence data [S. cerevisiae [Schizosaccharomyces pombe and the human yeast-like pathogens Candida albicans and C. neoformans. In this review, we aimed to describe the identification and classification of ZNFs and summarize the current state of knowledge of ZNFs in the sexual reproduction, virulence factor production, metal homeostasis, pathogenesis, and stress resistance in the human fungal pathogen C. neoformans.ZNFs are zinc finger domain-containing proteins and form the most abundant proteins, serving extraordinarily diverse biological functions, such as transcriptional activation and regulation, protein degradation, signal transduction, and plenty of other functions . Fungal nce data . Among trevisiae ,23. ZNFsXenopus laevis [ZNFs are wildly present in eukaryotes and participate in various cellular processes, including signaling transduction, gene activation and regulation, and cell differentiation. ZNFs are considered to be one group of proteins for they all harbor a common zinc finger motif, yet they display variable secondary structures and enormous functional diversity. A zinc finger is usually defined as a small, functional protein, with a structural motif that is coordinated by one or more zinc ions, and typically serves as an interactor module that binds DNA, RNA, proteins, or small molecules . The zins laevis  and it ws laevis . Since ts laevis . Howevers laevis ,22. 2H2, C2HC, C3H, C4, C6, C3HC4, C2HC5, C4HC3, and C8, in which C and H represent cysteine and histidine, respectively [2H2-type finger, represented by TFIIIA [2HC-type zinc finger domains, also referred to as retroviral-type (RT) zinc finger sequences, are required for viral genome packaging and RNA or single-stranded DNA binding in eukaryotes [3HC4-type finger, also termed RING finger, can be categorized into seven types with different conserved motifs, such as RING-H2, RING-HC, RING-v, RING-D, RING-S/T, RING-G and RING-C2 [2HC5 motif, also referred to as LIM, was first identified in the cell lineage gene lin-11 in Caenorhabditis elegans [ZNFs can coordinate divalent zinc ions with the combination of cysteine and histidine residues. Initially, based on the structural and functional variation of the zinc finger domain, ZNFs were categorized into at least nine types: Cectively ,27,28. Ty TFIIIA , is the y TFIIIA . C2HC-tykaryotes ,31,32,33karyotes ,34,35. Tkaryotes . GATA-1 karyotes . C3HC4-t RING-C2 ,39,40. M RING-C2 ,42. The  elegans .Then, the ZNFs were categorized into eight-fold groups based on the structural properties in the folded domain by a more systematic method . Among tC. neoformans has become the main fungal pathogen in individuals with impaired immunity [C. neoformans, the Cryptococcus research community has sequenced the genomes and/or transcriptomes of the two varieties of C. neoformans: C. neoformans var. neoformans  and C. neoformans var. grubii  [Cryptococcus genome contains a large number of genes annotated to encode ZNFs. When we performed a keyword search combined with “zinc finger” or “zinc-finger”, 127 and 129 genes encoding ZNFs were identified and characterized, according to the combination of cysteines and histidines, which coordinated Zn+2 ions in C. neoformans var. neoformans and C. neoformans var. grubii genome database, respectively  , providively See . Based oties see . The funC. neoformans, were identified and characterized via genetic and genomic approaches [ZNFs form one of the largest families of transcriptional regulators, playing an essential role in the growth and development in eukaryotes. Over the past two decades, multiple signaling pathways, such as pheromone-sensing, light-sensing, cAMP/protein kinase A(PKA), calcineurin, and high-osmolarity glycerol (HOG) signaling pathways, regulating sexual reproduction and hyphal morphogenesis in proaches ,55,56,57C. neoformans can undergo a dimorphic transition to a filamentous growth form by mating and monokaryotic fruiting  MAPK (mitogen-activated protein kinase) signal transduction cascade is a highly-conserved signaling circuit controlling the dimorphism transition during bisexual and unisexual reproduction [ZNF1α, located in the mating type locus, Znf2, and Znf3, residing in another genomic region [Cryptococcus dimorphic hyphal growth because Znf1 mutations in both mating types in the var. neoformans strain backgrounds did not suppress cell fusion or hyphal formation during bisexual and unisexual mating [2H2 zinc finger domains, indicating that it may be a transcription factor. Loss of Znf2 locks cells in the yeast phase, while overexpression of this regulator drives hyphal growth, indicating that Znf2 is critical for filamentation and hyphal morphogenesis after the cell-cell fusion event during mating [2H2 zinc finger domains and sexual reproduction analysis showed that Znf3 plays an essential role in both unisexual and bisexual reproduction [Cryptococcus pathogenic species [As a heterothallic basidiomycete, fruiting . Cell fuoduction . Howeverc region ,59,60. Fl mating . Proteing mating ,61,62,63g mating . Znf3 co species .C. neoformans mating, the resulting dikaryotic cell initiates filamentous growth, with two parental nuclei migrating coordinately in the hyphae. During monokaryotic fruiting, two cells of the same mating type, e.g., α cells, fuse to become diploid α/α cells to form the diploid monokaryotic hyphae and initiate filamentous growth. After fusion of the cells of the opposite mating types during C. neoformans. Calcineurin is a highly-conserved Ca2+/calmodulin-dependent protein phosphatase and involves stress responses, fungal morphogenesis, and virulence in the three major human fungal pathogens [C. neoformans [C. neoformans. Fu et al. demonstrated that Crz1 is essential for the sporulation process although it is not required for yeast-hyphal morphological transition during unisexual reproduction of C. neoformans [C. neoformans [The calcineurin signaling pathway and light-sensing pathway are involved in the regulation of hyphae growth in athogens ,67. Althoformans . Crz1 in light sensing were identified and named independently as CWC1/BWC1 (Cryptococcus WC-1 homologue/Basidiomycete White Collar 1) and CWC2/BWC2 (Cryptococcus WC-2 homologue/Basidiomycete White Collar 2) [BWC1 and BWC2 are essential for mediating light inhibition of mating and haploid fruiting [C. neoformans [Light inhibits mating and monokaryotic fruiting of ue light ,71. C. nollar 2) ,71, and ollar 2) . Functiofruiting ,71 and toformans . Yeh et oformans . C. neoformans mating or monokaryotic fruiting, a basidium formed at the tip of the mating hyphae and four chains of basidiospores are produced on top of the basidium. Signaling pathways, such as the MAPK pathway, ubiquitin-proteasome pathway, and quorum-sensing pathway, are involved in the regulation of basidiospore formation in C. neoformans. In the late stage of S. cerevisiae, providing a framework for studying morphogenesis in a variety of fungal species. STE12 (Sterile gene 12) encodes a downstream C2H2-type transcription factor of the pheromone response MAPK cascade, controlling fungal development in S. cerevisiae [STE12 homologs, STE12α and STE12a, have also been identified in C. neoformans and are encoded by the mating-type locus [STE12α or STE12a does not abolish pheromone sensing or mating, deletion of the gene results in defective monokaryotic fruiting in C. neoformans [The MAPK cascade, also referred to as the pheromone response pathway, regulates the dimorphic switch in revisiae . STE12 hpe locus ,75,76. Aoformans ,75,77. C. neoformans is essential for fungal sexual reproduction as basidiospore production was blocked in bilateral mating between fbp1Δ mutants, even though normal dikaryotic hyphae were observed during mating [2H2-type zinc finger protein (Zfp1) was identified as a potential substrate of Fbp1 by IP pulldown and LC-MS/MS methods. Functional analysis showed that Zfp1 is also essential for fungal sexual reproduction as deletion or overexpression of Zfp1 abolishes meiotic sporulation of C. neoformans, which indicates Zfp1 is essential for regulating meiosis during mating [F-box proteins are the key recognition subunit of the E3 ligase complexes that take part in protein ubiquitination and degradation . The F-bg mating . One C2Hg mating . C. neoformans [2H2-type zinc finger regulator (Cqs2) as a critical component of the Qsp1 signaling cascade during both bisexual and unisexual reproduction. Loss of Cqs2 significantly impaired bisexual and unisexual filamentation and abolished meiotic sporulation during both bisexual and unisexual reproduction, indicating ZNFs may play a role in bridging sexual production and the perception of surrounding stresses [Bacterial quorum sensing is a well-characterized cell-cell communication system to coordinate population density-dependent changes in behavior ,81. Receoformans . The autstresses . C. neoformans can infect the human CNS, causing meningitis and killing hundreds of thousands of people each year. Since it is a significant yeast pathogen with genetic tractability, extensive studies have been conducted on the pathogenesis in C. neoformans. Virulence factors and signaling pathways that are crucial for fungal virulence have been identified and extensively studied in C. neoformans [C. neoformans [oformans ,83,84,85oformans ,87,88,89C. neoformans has three classical virulence factors: melanin production, capsule formation and growth at 37 °C, which favors the infection and the pathogenesis of C. neoformans. Cryptococcus STE12α encodes a protein containing both homeodomain and zinc finger regions in MATα cells of C. neoformans [ste12α mutant was significantly reduced in a mouse model as deletion of STE12α resulted in a reduction in the capsular size of yeast cells in C. neoformans [STE12α reduces DNA binding ability, mating frequency, and haploid fruiting capability but increases virulence and capsule size of yeast cell in C. neoformans. In contrast, mutation in the zinc fingers region also resulted in virulence attenuation, capsule size reduction, and decreased gene expression of capsule associated genes [STE12 homolog, STE12a, was also identified in MATa cells in C. neoformans. Deletion of STE12a markedly reduced virulence in mice, as is the case with STE12α [STE12ap regulates mRNA levels of several genes that are important for virulence, including C. neoformans laccase gene (CNLAC1) and capsule (CAP) genes. These results clearly showed that mating-type specific genes are not only crucial for sexual reproduction but also play an essential role in virulence of Cryptococcus in host tissue.As a human fungal pathogen, oformans . Virulenoformans ,77. Mutaed genes . The sech STE12α . NortherC. neoformans, mutation of Pkc1 results in defects of cell wall integrity, aberrant capsule, decreased melanogenesis, and inability to grow at 37 °C [SP1 in a pkc1Δ mutant rescued the phenotype defects involved in virulence, including cell wall integrity, nitrosative stress, and extracellular capsule production. Besides, deletion of sp1 showed similar phenotype as pkc1Δ mutant, suggesting that the C-terminal ZNFs may shift from calcineurin-dependent to Pkc1-dependent transcription factors in the process of fungal evolution [Protein kinase C-1 (Pkc1) signaling pathways is the primary cellular pathway mediating the cellular responses to environmental stresses. In at 37 °C . Overexpvolution . C. neoformans can invade the CNS to cause cryptococcal meningoencephalitis, most commonly in individuals with impaired immunity. However, the molecular mechanism used by C. neoformans to invade the CNS is not very clear. Vu et al. identified a novel secreted metalloprotease (Mpr1) and demonstrated that it is required for C. neoformans to establish fungal disease in the CNS [Cryptococcus to cross the blood-brain barrier (BBB) by facilitating attachment of cryptococci to the endothelium surface. Strikingly, the sole expression of cryptococcal MPR1 in S. cerevisiae endowed the yeast cells with the ability to cross the BBB, which demonstrated that Mpr1 is specific in breaching the BBB and suggested that Mpr1 may function independently of the hyaluronic acid-CD44 pathway in breaching the brain endothelium [ the CNS . Mpr1 beothelium . Furtherothelium . The proothelium .C. neoformans in regulating the elaboration of the polysaccharide capsule during infection. Iron-responsive transcription factor (Cir1) is a GATA-type zinc finger protein regulating iron uptake, iron homeostasis, and virulence factor expression in C. neoformans. Mutation of Cir1 results in the loss of capsule production, decreased cell wall integrity and membrane functions, marked growth defect at host body temperature, increased activity of laccase, and complete loss of virulence in both serotype A and D strains in C. neoformans [Cryptococcus can perceive iron as part of the disease process through the zinc finger protein Cir1, which may provide opportunities for antifungal treatment.Metals are essential for life and play a central role in the struggle between microbial pathogens and their hosts . The uptoformans . Furtheroformans . Thus, C2H2-type zinc finger protein Zap1, an ortholog of ZafA and Zap1p in A. fumigatus and S. cerevisiae [C. gattii. Disruption of Zap1 not only impaired C. gattii growth in zinc-limiting conditions but also decreased its virulence in a murine model of pulmonary infection, indicating ZNFs is required for proper zinc metabolism and plays an essential role in cryptococcal virulence [The regulation of zinc homeostasis is vital for fungal survival and virulence . To inverevisiae ,95, was irulence .C. neoformans is confronted with a variety of host-specific stresses, such as nutrient limitation, temperature shift, changes in oxygen/carbon dioxide levels, oxidative/nitrosative stress, and osmotic stress, upon its infection of the host lung. Signaling pathways or factors involved in the stress response have been identified and extensively studied [C. neoformans.  studied ,97,98. ZC. neoformans serotype D strain. In contrast, Crz1 is also essential for cell wall damage repair, biofilm formation, and susceptibility to fluconazole in C. neoformans serotype A strain [As a calcineurin-responsive zinc finger transcription factor, Crz1 has been proved to govern unisexual reproduction in self-filamentous A strain . MoreoveA strain .S. cerevisiae, the C2H2-type ZNF Mig1 (multicopy inhibitor of GAL gene expression) is a carbon catabolite repressor responsible for the repression of genes involved in alternative carbon metabolism, respiration, and gluconeogenesis [C. neoformans is involved in the regulation of mitochondrial functions, such as respiration, tolerance for reactive oxygen species (ROS), and expression of genes involved in iron consumption and acquisition [MIG1 deletion increased fluconazole susceptibility in C. neoformans, highlighting an association between drug susceptibility and mitochondrial dysfunction and providing possible new targets for antifungal drug development [In ogenesis ,101. In uisition . Remarkaelopment .C. neoformans [The zinc finger protein Zfp1 is a potential substrate of the F-box protein Fbp1 and is involved in fungal sexual reproduction and virulence in oformans . Stress oformans .C. neoformans. Two small zinc knuckle RNA binding proteins, gluconeogenic growth suppressor 2 (Gis2) and zinc-finger protein 9 (Znf9), were identified by mass spectrometry in C. neoformans. Loss of Gis2 or Znf9 not only increased susceptibility to cobalt chloride, fluconazole, and oxidative stress but also repressed the levels of sterols in gis2Δ or gis2Δ znf9Δ double mutants although transcriptional induction of C-4 sterol methyl oxidase gene (ERG25) was similar to that of the wild type [C. neoformans.mRNA reprogramming is necessary for successful stress adaptation in ild type . These rThe ZNFs described in the literature and discussed in this review are summarized in C. neoformans. In this review, the various roles of ZNFs in C. neoformans were summarized.ZNFs are one of the most abundant groups of proteins and constitute the largest family of transcription factors that play important roles in prokaryotes and eukaryotes. Recent studies have shown that ZNFs play crucial roles in fungal development and differentiation, ion homeostasis, cell wall integrity, drug susceptibility, and virulence in the human fungal pathogen C. neoformans, there are still some areas that should be studied: first, the molecular mechanisms that link ZNFs to their biological roles; second, only 20 ZNFs have been identified and functionally analyzed in C. neoformans; third, most of the ZNFs that have been functionally characterized in C. neoformans belong to the C2H2 family; and fourth, although many studies have investigated the role of ZNFs in resistance to antifungal drugs, new antifungal drug development is still an issue. Thus, it is also urgent to investigate the molecular basis of ZNFs in the interaction between C. neoformans and host cells since most ZNFs are involved in fungal virulence in C. neoformans. At last, it will be of great interest in the future to see whether some ZNFs could be a target to develop new antifungal drugs.Although many ZNFs have been studied and have been shown to play important roles in fungal development, stress response, and virulence in


Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a “calm” or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a “activated” state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells. Extracellular nucleotides  are emerging as important autocrine/paracrine regulators of the vascular, immune, and hematopoietic systems ,7,8,9,10The wide distribution of purinergic receptors in the vascular system, together with the expression of multiple receptor subtypes in endothelial cells, point to their physiological and pathophysiological importance. It is also becoming evident that alterations in the purinergic signaling may result in the development of a variety of neurogenerative, immune, and vascular pathologies ,10,18. S2, extracellular adenosine and others. As an example of stepwise activation, P2Y2 activity is most effective at stimulating EC proliferation when working in conjunction with other growth factors or cytokines [The vascular endothelium is a semi-selective diffusion barrier between the plasma and interstitial fluid and is critical for normal vessel wall homeostasis. In addition, due to its secretory and adhesive properties, the endothelium is recognized as an active part of the vasculature ,39. In tytokines . Other eytokines . There i2+ pathways in the endothelium, in turn leading to eNOS-NO-mediated vasodilation [2+-dependent [2+ increased-sparks in the vicinity of TRPV4-GPCR, leading to localized eNOS activation through the calmodulin-binding domain [Vasodilation occurs via multiple mechanisms and is protective against high blood pressure, a major pathological factor in cardiovascular disease (CVD) . Fluid sdilation . Mice widilation . Endothedilation . The smaependent . Activatg domain ,44.2 [Mechanisms contributing to decreases in blood pressure are mediated by the activation of P2Y2. . Interme2 .Experimental evidence points to the involvement of P2YRs in aging-dependent hypertension . Aging hType 2 diabetes (T2D) is known to impair vascular perfusion through poorly defined mechanisms. It was found that diabetics have impaired vasodilator responses to purinergic, but not to muscarinic or nicotinic receptor-induced vasodilation, similar to the situation in aging . In the Oxidative stress is a feature of many CVDs and often closely correlates with inflammation, as both professional phagocytes bound to ECs and ECs activated by cytokines/TLRs produce ROS . P2Y1 stInflammation is a characteristic feature of CVD and is mediated by released cytokines, chemokines, eicosanoids, and ATP by vascular and blood cells. ECs represent a primary cellular target for many pro-inflammatory stimuli. Potent pro-inflammatory mediators, such as LPS and TNFα, significantly induce the P2Y6 receptor expression in EC in vitro and in vivo . In vascThe P2Y11 receptor, activated by ATP, has anti-inflammatory actions, which could be implicated in EC protection in CVDs . In ECs,The mechanism by which P2Y11 exerts anti-inflammatory action are not known, but P2Y11 is a dual-specificity GPCR, activating both Gq and Gs. The activation of Gs has been reported to be potentially more circumstance- or cell-type-dependent . HoweverP2Y11 receptor has demonstrated anti-inflammatory effects on ECs in other settings, such as in tumors, where ECs often migrate from the tumor locus. In this setting, cytokine- growth factor-, and chemokine-driven migration can be inhibited by activation of P2Y11. Such effects require adenylate cyclase 10 isoforms of the enzyme ,76. In aIn atherosclerosis, the P2Y2 receptor apparently has pro-inflammatory actions, as ApoE-/-mice with additional deletion of P2Y2 have lower inflammatory indices in an atherosclerotic lesion, which includes lower VCAM-1 and LT-α, a member of TNFα family known to be pathogenic in the disease . As suchActivation of endothelial P2Y2 also has been linked to the mechanism by which oxidized LDL promotes vascular inflammation in atherosclerosis. P2Y2 up-regulates cell adhesion molecules such as VCAM-1 and ICAM-1 to secure monocyte adherence and infiltration into the atherosclerotic lesion . P2Y6 alAdditional evidence suggests that GPR120, a GPCR for omega-3 fatty acid, seems to counteract oxidized-LDL/P2Y2-mediated inflammation in atherosclerosis, inhibiting the binding of monocytes to ECs . GPR120 It is thought that in a hyper-adiposity state associated with metabolic syndrome, a high turnover of adipocytes is observed despite increased fat content. Death of adipocytes promotes sterile inflammation, and this is the connection to CVD. ATP, being released from EC and other cell type, is pro-inflammatory and instigates leukocyte infiltration via P2Y2 receptors. P2Y2-null mice show blunted responses on a high-fat diet, gain less weight, and have better insulin sensitivity and lower cholesterol levels . AdiposeIncreased vascular permeability is an important pathophysiological component of many CVD and lung diseases, including acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), which arise from a wide range of lung injuries such as toxins or inflammatory mediators, resulting in significant morbidity and frequently in death ,88,89. IIn addition to the regulation of vascular tone, P2Y1,2,4,6 receptors seem to potentiate vascular inflammation and increase vascular leak, decreasing the barrier function of the endothelium . It has 2 expressed at the highest and Gαi1 at the lowest  levels , an inverse permeability index  levels A. Specifty index B confirm in vivo . While t in vivo . In this in vivo ,101. Impgulation .+ emerges as an adenine nucleotide that can be released from neurons and blood vessels spontaneously and by regulated mechanisms [+ has also been recognized as an extracellular signaling molecule involved in cell-to-cell communication [Nicotinamide adenine dinucleotide (NAD) is a cofactor that is central to metabolism. In addition to its metabolic functions, NADchanisms ,103. In nication ,104,105.nication . In addinication ,36. In anication .Consistent with the in vitro studies, β-NAD attenuated the LPS-induced inflammatory cells infiltration and decreased permeability in the lungs of LPS/β-NAD-treated mice compared to mice treated with LPS alone. Histological specimens from the lung showed increased, and prominent interstitial edema with infiltration of neutrophils in the lung parenchyma of mice treaded with LPS alone compared to the LPS/β-NAD-treated lungs . TherefoPurinergic regulation of vascular growth and development is a rapidly developing concept in vascular biology, focusing on the role of specific P2YR subtypes. In addition to vasoprotective vasodilation, P2Y receptors are critical to angiogenesis, vasculogenesis, and endothelial differentiation. Several cell and animal studies have provided evidence on the importance of nucleotide signaling for vascular growth. A study on P2Y4 knockout mice demonstrated a role for this receptor in cardiac micro-vessel growth, migration, and PDGF-B secretion in response to UTP. This study also showed that P2Y4 is an essential regulator of endothelial-cardiomyocyte interactions in post-natal heart development . DecreasVasa vasorum (VV) is the microcirculatory network of the bronchial (systemic) circulation and, similar to its role in systemic vessels, contributes to vascular integrity through the supply of oxygen and nutrients to the outer part of the pulmonary artery (PA) wall. Studies on animal models of PH revealed marked adventitial thickening and expansion of the VV network, which are especially prominent components of the pulmonary vascular remodeling process [Proliferative vascular remodeling has a central role in the pathology of many cardiovascular diseases ,115,116. process ,118. An  process ,124,125. process . At pres2+, [The findings from a neonatal bovine model of PH demonstrated that ATP is a potent angiogenic factor for PA VVEC ,127. Mor2+, ,137,138.2+, . Therefo2+, ,127,139.As indicated above, pulmonary artery VV angiogenic expansion is a characteristic feature of hypoxia-induced pulmonary vascular remodeling in PH. The expression of P2Y13 has been previously shown in spleen, brain, and circulating CD34+ progenitor cells, but not in vascular endothelium ,140. TheInfantile hemangiomas are vascular tumors characterized by excessive proliferative/dysregulated endothelial proliferation and spontaneous involution. However, the mechanisms underlying their unique “lifecycle” remain largely unknown. Previous studies on hemangiomas have been restricted by elucidating canonical angiogenic pathways that include vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2, IGF-I, Tie-1, Tie-2, bFGF, and angiopoietin . ConsideEndothelial purinergic signaling in CVD remains the area of intensive investigation. With the growing interest in pathological vascular remodeling, inflammation, angiogenesis, acute lung injury, stem cell differentiation, and vascular tumors, the regulatory circuits of extracellular nucleotides that operate via the activation of purinergic receptors may bring a new dimension to a better understanding of CVD mechanisms . Researc


The term “inflammaging” describes the chronic, low-grade systemic inflammation that occurs during physiological aging in the absence of an overt infection ,2. InflaMir34a deletion in mice reduces the expression of the VC markers such as SRY (sex-determining region Y)-box 9 (Sox9) and Runx2 and senescence proteins p16 and p21 and, consequently soft tissue and aorta medial calcification [MicroRNA-34a (miRNA-34a) is a senescent-associated miRNA whose expression has been shown to increase in different tissues and organs with age ,10. miR-Mir34a genetic ablation prevents the induction of IL6 expression occurring during aortas medial calcification onset. Importantly, we also found a positive correlation between circulating miR-34a and IL6 in a population of healthy subjects spanning from 20–90 years [Our recent work, published in the Special Issue “Mechanisms of Inflammation in Degenerative Cardiovascular Conditions 2.0” of this journal, demonstrates that miR-34a enhances the secretion of several SASP factors in HASMCs, such as pro-inflammatory molecules ), the metalloprotease inhibitor TIMP2 and the Insulin-like Growth Factor Binding Protein 3 (IGFBP3) . Precond90 years . Altoget+ stem cell differentiation, a cell population endowed of both regenerative and hematopoietic properties, towards more inflammatory cell populations [T2DM shares a number of important features with aging, including inflammaging and VC ,13. Indeulations . To thisulations ,17,18. Iulations . In the ulations . In partulations . These culations , with clulations ; howeverSince miRNAs are emerging as promising druggable targets, extending the knowledge of the mechanisms by which miR-34a regulates cell senescence, trans-differentiation and SASP acquisition in different pathological contexts, will help to develop new pharmacological therapies to counteract inflammaging and, eventually, age-related diseases onset.


MYC, BCL2, and/or BCL6 gene rearrangements. While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, and their contributions to rearrangements have yet to be fully elucidated. Using FISH and high-resolution CNA data, we defined the landscape of concurrent gene rearrangements and copy gains in MYC, BCL2, and BCL6, in a cohort of 479 newly diagnosed DLBCL. We also show that concurrent translocations and copy number alterations, in combinations similar to DH/TH, identify a unique subset of DLBCL, alternative DH/TH, that have survival outcomes similar to DH/TH DLBCL patients.Double/triple hit lymphoma (DH/TH), known as high-grade B-cell lymphoma (HGBL), is an aggressive diffuse large B cell lymphoma (DLBCL), defined as having concurrent  Factors that predict DLBCL prognosis include clinical characteristics3, cell-of-origin (COO)5, MYC translocation status8, and genomic alterations11. Concurrent rearrangements of c-MYC and BCL2 and/or BCL6 detected by fluorescence in situ hybridization (FISH), also known as “double hits” (DH), occur with a frequency of 6–14%12, are associated with transcriptional dysregulation of MYC, and have been correlated with poor prognosis in patients treated with chemotherapy alone or with the addition of rituximab14. In the revised fourth edition of the World Health Organization (WHO) classification of lymphoid neoplasms published in 201615, DH/triple-hit (TH) DLBCL was reclassified as high-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements. In addition, double-expressor lymphoma, defined as overexpression of MYC and BCL2 proteins without chromosomal rearrangements, although not a distinct WHO classification, accounts for 20–30% of DLBCL cases and also has poor outcomes13.Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive non-Hodgkin lymphoma subtype in Western countriesMYC, BCL2, and BCL6 represent significant genomic events in lymphoma, less is known about the contribution of chromosomal copy number alterations (CNAs) in these genes to existing translocations. MYC copy gains are detectable in DLBCL and have been shown to increase MYC mRNA expression in DLBCL19. Likewise, MYC translocation negative patients have been shown to have MYC IHC scoring similar to those harboring the translocation, alluding to alternative mechanisms driving MYC expression in DLBCL20. While the DH/TH cases have concurrent rearrangement events in MYC, BCL2, and BCL6, it is not clear whether an expanded definition could include copy number gains as a secondary event. To date, two studies have examined the impact of both translocations and CNAs, occurring intrans, in MYC, BCL2, and/or BCL6, detected by FISH. Quesada et al. and Li et al. both identified atypical DH/TH cases in large cohorts, with concurrent MYC and BCL2 and/or BCL6 abnormalities, other than double translocation, which had outcomes similar to bona fide DH/TH. However, several limitations exist in regards to the analysis within these studies, including limited definition, BCL6 exclusion, case selection, and/or copy number detection method21. Lu et al.22 similarly suggested that MYC and BCL2 double CNA (in cis) constituted a group of high-risk DLBCL, though MYC and BCL2 were never analyzed in combination with FISH, as in the previous studies. Likewise, two of the aforementioned studies acknowledge BCL6 as a defining gene for DH/TH lymphoma, yet neither included it in their analysis. Understandably, it is very difficult to acquire data on enough patients to power investigations into relatively rare subtypes, such as these. These studies, in addition to many others, rely on the existing clinical FISH data to roughly determine gene-specific CNAs. High-resolution copy number analyses, such as copy number arrays, are not routinely performed on DLBCL patients and are not commonly available. Therefore, it remains to be defined whether or not translocations and copy number events in MYC, BCL2, and BCL6 cooperate to identify new DH-like or atypical-DH lymphoma groups. In addition, the relative prevalence of these cases in the DLBCL population and the clinical outcome of these cases is yet to be fully explored. In this study, we define the landscape of MYC, BCL2, and BCL6 translocations in combination with CNAs in two independent cohorts of DLBCL and address the key question of how CNAs impact DLBCL prognosis.While translocations in n = 260) were recruited and prospectively followed through the Molecular Epidemiology Resource (MER) of the University of Iowa/Mayo Clinic Lymphoma Specialized Program of Research Excellence. Full details of this prospective cohort study of lymphoma outcomes have been previously published23. This study was approved by the Mayo Clinic Institutional Review Board. This analysis was restricted to DLBCL patients treated with frontline immunochemotherapy and who had available FISH and CNV data. COO on available MER cases was determined by gene expression profiling (n = 15)24, Lymph2Cx assay 25, or the Hans algorithm (n = 110)26. An additional 219 DLBCL cases were included from Chapuy et al.9 and were acquired from the journal website Supplementary Data Tables For this study, newly diagnosed and consented DLBCL patients n = 60 were rling n = 524, LympMYC (8q24.1), BCL2 (18q21), and BCL6 (3q27) were determined by FISH break-apart probes  on tissue microarray (TMA) slides, according to standard protocols. Briefly, TMAs were deparaffinized, renatured, and dehydrated. Probe working solutions were mixed using 1 μL of the concentrated probe  with 9 μL of LSI/WCP® hybridization buffer (Abbott Laboratories). The working solutions of BAP BCL6(3q27), BAP MYC(8q24.1), and BCL2(18q21) were applied to the target areas, coverslipped, co-denatured with a ThermoBrite™ at 83 °C for 5 min, and hybridized overnight in a 37 °C humidified oven. Following hybridization, slides were soaked in room temperature (RT) 2×SSC/0.1% NP-40 to remove coverslips, placed in 2×SSC/0.1% NP-40 at 74 °C for 2 min and then placed into RT 2×SSC/0.1% NP-40 for 2 min. The slides were stained with 4′-6,-diamidino-2-phenylindole (Vector Laboratories) and coverslipped. The slides were analyzed by a technologist using standard fluorescence microscopy methods. Tissue samples were scanned in their entirety, and the qualitative result was determined based on observed signal patterns. Visualization of the FISH signals was accomplished by using the fluorescence microscope, and pictures were captured by using a FISH imaging system .For the Mayo Cohort, translocation events in 27. Briefly, DNA, extracted from formalin-fixed, paraffin-embedded (FFPE) DLBCL tumors (n = 247), was processed at the Mayo Clinic Cytogenetics Lab using the molecular inversion probe OncoScanTM FFPE Assay Kit . For an additional 13 cases, whole-exome sequencing files were used. Raw WES bam files and OncoScan OSCHP files were analyzed using Nexus Copy Number 9.0 software . Data interpretation and copy number calling were made using the human reference genome GRCh37/hg19. Nexus standard configuration for gain calling thresholds was used. All gains and high-copy-gains (amplification) are reported in this study as copy number gains. Copy number losses were excluded from this analysis.Methods for analysis of copy number, including sample collection, analysis, and data interpretation has been previously published9. Presence or absence of MYC, BCL2, and BCL6 structural variants was reported from sequencing data as 0 (Absence), and 1 or 3 (presence). Copy number gains were reported as 0 (Absent), and 1 or 2 (Present) for MYC (8q24.22), BCL2 (18q/18q21.33), and BCL6 (3q/3q28). Both arm length and q-band gains/amplifications were considered for BCL2 and BCL6.Gene-specific structural variants and copy number gains were obtained from Supplementary Table Positive protein expression was assayed by immunohistochemistry in the Mayo Clinic Pathology Research Core using standard MYC, BCL2, and BCL6 clinical antibodies. Positivity was determined according to published methods: MYC-IHC+ (≥40% of cells), BCL2-IHC+ (≥50% of cells), and BCL6-IHC+ (≥30% of cells). Protein expression was completed in 192 samples for MYC, 201 samples for BCL2, and 41 samples for BCL6. Scoring was completed by a hematopathologist in the Mayo Clinic Department of Lab Medicine and Pathology.www.graphpad.com. Survival data were analyzed using a cox-proportional-hazards model within the Survminer package in R29. Sensitivity analysis was used to adjust the model for single covariates, one at a time, within key clinical subsets of patients within the cohort. All hazard ratios (HR), 95% confidence intervals, and Kaplan–Meier estimates were generated from the package.Chi-square test-of-independence was performed using GraphPad Prism version 8.1.1 for Windows, GraphPad Software, La Jolla, CA, USA, MYC and BCL2 and/or BCL6, FISH and copy number data from 260 DLBCL patients, from the Mayo/Iowa MER (Mayo Cohort), were analyzed. The analysis flow for the study is shown in Fig. MYC, BCL2, or BCL6. This identified three groups: DH/TH cases; those with one or more translocations, but not defined as DH/TH ; and those with no translocations (translocation negative). We next evaluated copy number gains, henceforth referred to as CNA, in MYC, BCL2, and BCL6 in the same patients using OncoScan Array. In this analysis, translocation-negative cases were broken into those with copy number gains (CNA positive) and those without translocations or copy number gains . From the translocation-positive cohort, we identified a subset of patients that had concurrent events in MYC and BCL2 and/or BCL6, but a translocation in one gene and a CNA in another, distinguishing a subset of a non-canonically defined DH/TH . The results from this analysis highlight a complex landscape of translocation and CNAs , in which structural variants and copy number gains in MYC and BCL2 and/or BCL6 were identified  identified 21 (4.4%) DH/TH cases, 51 (10.6%) Alt-DH/TH cases, 147 (30.7%) Tx positive cases, 133 (27.8%) CNA positive cases, and 127 (26.5%) with no alterations. A summary of the number of cases in each subgroup and the clinical characteristics of each cohort is shown in Supplemental Table MYC translocations have concurrent BCL2 and/or BCL6 gains . MYC, BCL2, and BCL6 positivity were defined as an expression by ≥40%, ≥50%, and ≥30% of malignant cells, respectively, in accordance with established cutoffs13. Positive MYC expression was detected in 81.0% of MYC translocation cases and 14.3% of MYC CNA cases, suggesting that only a small subset of MYC CNAs result in elevated protein expression , a majority (56%) were found in GCB cases. However, the addition of copy number data yields a much different story, where activated B cell-like (ABC) cases were found to be enriched for copy number gains in MYC, BCL2, and BCL6  cell COO subtypes typically resulting from CL6 Fig. . Given t31, little is known about the contribution of CNAs, in particular, the impact on patient outcomes. Therefore, we compared the clinical characteristics and outcome of Alt-DH/TH cases with DH/TH and all other groups combined. The survival curves for translocation-positive, copy-gain-positive, and no alterations, and event combinations within these groups, were overlapping and statistically indistinguishable; we, therefore, combined them and considered this combined group as controls (Other) for statistical calculations . Because of the increased prevalence of ABC cases in the Alt-DH group, compared to DH/TH, we performed a secondary analysis based on COO  and OS of all three groups. The Kaplan–Meier curves for EFS/PFS (upper panel) and OS (lower panel), between DH/TH, Alt-DH/TH and Other, are significantly different  as reference, we observed significant HRs for EFS/PFS in the DH/TH group and HRs suggestive of poor OS in both DH/TH and Alt-DH/TH . Consistent with our previous result, ABC-like Alt-DH/TH (n = 15) cases had significantly poor HRs for EFS/PFS and OS , when compared to ABC-like Other cases (n = 151) as reference. The outcomes were consistent within each cohort and remained consistent when adjusting for age and cohort, independently, in a multivariate model . Using Other DLBCL  independent of DH/TH translocation status. Our work also corroborates prior studies, as many previous definitions of Alt-DH/TH share similar features and outcomes with this study21. However, this study includes an expanded definition, with BCL6 translocations and copy number events, and utilized high-resolution copy number analysis, which was not included in prior studies. Interestingly, MYC copy gains were not correlated with positive expression by IHC, unlike BCL2 and BCL6. Despite this, negative outcomes were seen in Alt-DH/TH patients of ABC-like COO, which includes most of the BCL6-Tx/MYC-CNA cases. Furthermore, our work complements recent results from Hilton et al.33 that identify double hit gene signature positive patients with alternative genomic events, cryptic to standard FISH, affecting MYC, BCL2, and downstream pathways.We have described the landscape of concurrent copy number gains and translocations in The proportion of Alt-DH/TH within all ABC-like cases in this study (8.8%) was similar to the proportion of DH/TH within all GCB cases in this study (7.8%) and therefore could be important for future screening approaches. Ongoing studies may further connect additional somatic events which correlate with the ABC-like Alt-DH/TH and further explain the poor clinical outcome of these patients. However, based on our existing results, we can conclude that Alt-DH/TH patients with ABC-like COO are a particularly aggressive subgroup of DLBCL and have outcomes resembling that of canonical DH/TH patients. Moving forward, it will be important to independently validate our findings on the impact of Alt-DH/TH on DLBCL outcome and consider assessing CNAs clinically.Supplemental Figures


FMR1) and cytoplasmic fragile-X mental retardation protein (FMRP)-interacting protein 1 (CYFIP1) contain 2 such examples of highly penetrant risk variants and encode synaptic proteins with shared functional significance. In this review, we discuss the biological actions of FMRP and CYFIP1, including their regulation of (i) protein synthesis and specifically FMRP targets, (ii) dendritic and spine morphology, and (iii) forms of synaptic plasticity such as long-term depression. We draw upon a range of preclinical studies that have used genetic dosage models of FMR1 and CYFIP1 to determine their biological function. In parallel, we discuss how clinical studies of fragile X syndrome or 15q11.2 deletion patients have informed our understanding of FMRP and CYFIP1, and highlight the latest psychiatric genomic findings that continue to implicate FMRP and CYFIP1. Lastly, we assess the current limitations in our understanding of FMRP and CYFIP1 biology and how they must be addressed before mechanism-led therapeutic strategies can be developed for psychiatric disorders.There is increasing awareness of the role genetic risk variants have in mediating vulnerability to psychiatric disorders such as schizophrenia and autism. Many of these risk variants encode synaptic proteins, influencing biological pathways of the postsynaptic density and, ultimately, synaptic plasticity. Fragile-X mental retardation 1 ( Neurotransmission between presynaptic and postsynaptic terminals is the adaptive communication mechanism linking neurons and other cell types into neural circuits and networks, which form the basis of synaptic plasticity, cognition, and behaviour , 2. The FMR1 gene (Xq27.3) [FMR1 mRNA is expressed in the neuronal cell body, developing and mature axons, dendrites and dendritic spines, and the nucleus [One such synaptic protein is fragile-X mental retardation protein (FMRP), encoded by the (Xq27.3)  and the (Xq27.3) . FMR1 mR nucleus , 15, but nucleus , 17. FMR nucleus , capable nucleus , 20, eit nucleus , 22.initiation by binding to the initiation factor eIF4E and FMRP-binding partner cytoplasmic FMRP-interacting protein 1 (CYFIP1)  [elongation stages of mRNA translation by stalling ribosomes on FMRP target transcripts [FMRP, its target mRNA, and other protein partners together form large messenger ribonucleoparticles (mRNPs) . Within ” later) , 32, alt” later) , 34, 35.nscripts , 24, altnscripts . FMRP isnscripts , 38. SomBeyond translational silencing, FMRP plays other biological roles , includiFMR1 can be better predicted. Such studies have used immunoprecipitation followed by either microarray [Considerable effort has been made to identify the mRNAs targeted by FMRP so that biological pathways affected by mutations in croarray , 51 or hcroarray , 52 to dcroarray . Therefocroarray  were alscroarray , 56, 57.Gene ontology analyses of brain-derived FMRP targets confirm an overrepresentation of genes involved in functions related to synaptic activity, plasticity, development, and anatomy , 52, 58,Whilst studies of FMRP targets have identified probable interactions between FMRP and ribosomal mRNAs, further work is needed to determine whether the translation of these mRNAs is indeed repressed by FMRP within the regulatory complex together with CYFIP1 and eIF4E proteins in the cell , 32.CYFIP1 is a highly dynamic synaptic protein involved in numerous biological pathways through an array of protein-protein interactions , Nck-associated protein 1 (NCKAP1), and haematopoietic stem/progenitor cell protein 300 (HPSC300) components . WithoutCyfip1 knockout (KO) mice, thought to derive from changes in presynaptic terminal size and enhanced vesicle release probability, and driven by dysregulation of the WAVE complex [cyfip1 mutant fly models that specifically found alterations in actin polymerization in presynaptic terminals [Cyfip1 KO mice were found to have decreased myelination of callosal axons, alongside impaired presynaptic neurotransmission in the corpus callosum [CYFIP1 belongs to the two aforementioned complexes, FMRP and WAVE, in a mutually exclusive manner, skewed towards greater association with the WAVE complex, under basal conditions . Notably complex . These ferminals , 80. Morcallosum .CYFIP2 mRNA is a target of FMRP [CYFIP1 is embryonically lethal [CYFIP1 has a closely related paralogue, CYFIP2, with over 88% amino acid identity . Like CY of FMRP , implyiny lethal , 84. Fury lethal .Considerable evidence suggests that a wide range of neuropsychiatric disorders, such as FXS, autism spectrum disorders (ASDs), schizophrenia, intellectual disabilities (IDs), and bipolar disorder, exhibit convergent synaptic pathology , 90, 91.In light of this view, and the biological importance of FMRP and CYFIP1 at the synapse , we will now consider the role of FMRP and CYFIP1 in the aetiology of psychiatric disorders, using data from human patient studies, especially psychiatric genomics, and preclinical models.FMR1 gene by a triplet repeat expansion  in the 5-untranslated region of FMR1[In humans, transcriptional silencing of the n of FMR1 leads ton of FMR1, 109. FXn of FMR1, 112, aln of FMR1, increasn of FMR1, 114, aln of FMR1, structun of FMR1, and a rn of FMR1, 119. Inn of FMR1 and the n of FMR1, 117. FMn of FMR1, 125.Fmr1 mutations have been interrogated preclinically for 25 years through the Fmr1 KO mouse model [Fmr1 KO rat model [Fmr1 KO mouse and rat models, especially in 3 key areas: dendritic spine maturation [The effects of se model  and, witat model , 128. Maturation , 130, elturation , 133, anturation , abnormaturation , social turation , and redturation , 141.Fmr1 KO rodents display a lack of mGluR-dependent translational control, which results in an elevated protein synthesis-dependent form of synaptic plasticity, known as mGluR-mediated long-term depression [Fmr1 KO phenotypes. The altered mGluR5 signalling in the absence of Fmr1 appears to be mediated through the preferential interaction of mGluR5 with activity-dependent isoforms of Homer1 over constitutive Homer proteins [In addition to heightened global protein synthesis, pression , 145. Inpression , or the pression , 146, caproteins , 148.Fmr1 results in the loss of the repressive eIF4E-Cyfip1-FMRP complex, which de-represses the initiation complex, eIF4F, required for cap-dependent translation initiation of FMRP targets [Fmr1 KO mice, and the restoration of this imbalance rescues spine and memory deficits in these animals [Fmr1 KO rodent model have illuminated a variety of molecular mechanisms relevant to FXS, especially those pertinent to the regulation of protein synthesis, and may provide biological targets for therapeutic intervention [The deletion of  targets . It was  animals . Hence, rvention , complemrvention , 152.FMR1 gene, a number of rare pathogenic point mutations have been reported that cause developmental delay and ID reminiscent of FXS [FXR2 gene might also contribute to ID [FXR1 gene confer risk to schizophrenia, bipolar disorder, and autism [FMR1 and psychiatric disorders derives from enrichment of association within the gene targets of FMRP (among which the fragile-X family genes themselves are included).Beyond repeat expansions in the t of FXS , 157. Fute to ID , 160. Whd autism , 165, thA set of FMRP target mRNAs derived from a study of mouse cortical polyribosomes  have beeBrain FMRP targets overlap considerably with other gene sets associated with psychiatric disorders, such as genes encoding PSD proteins and those involved in calcium signalling, synaptic plasticity, learning, and memory , 58, 181CYFIP1 is cytogenetically positioned in the non-imprinted 500-kb region between BP1 and BP2 on chromosome 15 , along with 3 additional genes: non-imprinted in Prader-Willi/Angelman 1 (NIPA1) and 2 (NIPA2), and tubulin gamma complex-associated protein (TUBGCP5) [The proximal long arm of human chromosome 15 (15q11.2–13.3) is a region of numerous low-copy repeats that can lead to aberrant meiotic chromosomal rearrangements. These result in deletions or duplications of sections of DNA, known as copy number variants (CNVs), and occur at any of 5 common breakpoints (BP1–BP5) on chromosome 15 . NeurodeTUBGCP5) . The 15qTUBGCP5) , 185. LaTUBGCP5) .Deletions or duplications of 15q11.2 are present in 1 of 100 people who present for genetic screening, whilst incidence in the general population is likely to be nearer 1 in 500 people . The CNVCYFIP1 is widely regarded as the most likely to confer the biological and behavioural phenotypes associated with 15q11.2 BP1–BP2 CNVs [CYFIP1, they are all expressed in the central nervous system and their expression is altered in patients with 15q11.2 CNVs [Among the genes located within the 15q11.2 locus, BP2 CNVs , 191. ThBP2 CNVs , 74. FurBP2 CNVs ; iPSCs dBP2 CNVs ; and theBP2 CNVs . However1.2 CNVs .Cyfip1 dosage through a variety of in vitro and in vivo rodent preclinical models. For instance, the heterozygous deletion of Cyfip1 in mice results in changes in dendritic and spine morphology [Cyfip1 also impinges on dendrite and spine morphology [Cyfip1 appears to affect protein synthesis under basal and activity-dependent conditions. The knockdown of Cyfip1 in cortical neurons in vitro increases the translation of FMRP target, activity-regulated cytoskeleton associated (ARC), under basal conditions and also ablates the activity-dependent translation of ARC, using brain-derived neurotrophic factor treatment to mimic synaptic activation [Cyfip1 heterozygous KO mice, whereby brain-derived neurotrophic factor treatment was insufficient to release the Cyfip1-FMRP complex from eIF4E, preventing the formation of the eIF4F complex, which subsequently prevented activity-dependent translation of ARC protein [Great strides have been made in understanding the consequences of altered rphology , 82, whirphology , whilst rphology . Meanwhitivation . Similar protein .Cyfip1 dosage have revealed elevated levels of mGluR-mediated long-term depression, which become disassociated from mRNA translation pathways [Fmr1 KO rodent models [Cyfip1 in CA1 hippocampal neurons can lead to increased excitatory neurotransmission, and a concomitant decrease in gamma aminobutyric acid (GABA)ergic neurotransmission at inhibitory synapses, shifting the overall excitation/inhibition balance towards excessive excitation [Cyfip1 in CA1 hippocampal neurons increased inhibitory GABAergic neurotransmission, along with increased expression of GABA receptors, suggesting a shift of excitation/inhibition balance towards greater inhibition [Cyfip1 mouse model, which better models the reduced dosage of CYFIP1 in 15q11.2 deletion patients, GABAergic signalling remains unaltered in the hippocampal dentate gyrus [Measures of synaptic plasticity in preclinical models of altered pathways  − findint models , 144. Ovcitation . The samhibition . Howeverte gyrus .Cyfip1 dosage. In Cyfip1 heterozygous KO mice, bilateral connectivity was shown to be reduced across multiple brain regions using resting-state functional magnetic resonance imaging [Cyfip1 haploinsufficiency [Cyfip1 dosage but increased in 15q11.2 deletion patients. This will require further study and may alter our current perception of the effect of CNVs of the 15q11.2 interval.Brain connectivity and white matter architecture appear to be especially sensitive to reduced  imaging . These aficiency . HoweverCyfip1 dosage also offer the chance to thoroughly assess changes in behaviour and cognition, prominent features in 15q11.2 deletion (and duplication) patients. Bozdagi and colleagues [Cyfip1 haploinsufficient mice, and found many aspects of spatial and fear learning and memory to be intact, with the exception of a rapid loss of extinction memory assessed using the inhibitory avoidance paradigm. Subsequent analysis of Cyfip1 heterozygous KO mice and rats has shown specific deficits in motor learning [Cyfip1 results in cellular phenotypes, particularly at the dendritic level [Cyfip1 in preclinical models leads to profound alterations in cellular and plasticity phenotypes, alongside mild behavioural phenotypes, many of which not only overlap with FXS and the Fmr1 KO model  in de novo CNV deletions from patients with schizophrenia [The relevance of CYFIP1 to schizophrenia becomes especially apparent when considered in the wider context of its biological actions within protein complexes. CYFIP1 is involved in the regulation of ARC protein and ARC-related genes, sometimes referred to as the “ARC complex” (a gene ontology-based complex). ophrenia . The genophrenia , 174 andophrenia , 179. FuFMRP and CYFIP1 are hubs for several biological pathways critical to synaptic plasticity. From preclinical models, we know that reduced expression of either CYFIP1 or FMRP results in a set of core phenotypes: altered spine and dendritic morphology, dysregulated protein synthesis, and elevated long-term depression. A further layer of complexity is added when it is considered that the concerted action of FMRP and CYFIP1, as part of the CYFIP-FMRP complex, represses the translation of hundreds of FMRP targets, likely influencing multiple downstream pathways. The importance of this system to synaptic function is recurrently highlighted by genetic studies demonstrating the risk conferred to psychiatric disorders by variants affecting genes encoding CYFIP1, FMRP, and their targets.Fmr1 and Cyfip1 deletion are only broadly similar, and in some cases diametrically opposed [Nevertheless, there are many questions that still surround the biology of FMRP, CYFIP1, and FMRP targets in health and disease. For example, whilst FMRP synaptic biology is well-characterized and preclinical techniques can reverse disorder-relevant phenotypes , 151, at opposed . These bCYFIP1 predispose carriers to increased risk for schizophrenia (mainly 15q11.2 deletions), autism, and ID (mainly 15q11.2 duplications), and likewise FMR1 deletions predispose carriers to autism and ID. These apparently pleiotropic effects might suggest that the categorical nature of diagnoses for psychiatric disorders needs to be fundamentally re-evaluated. Indeed, at the clinic, there are many common patient symptoms that span across diagnostic categories, and patients often present with comorbidities. The genomic findings point towards a continuum of causality, whereby common biological mechanisms, influenced by a range of convergent genetic factors, span across the traditional diagnostic boundaries of psychiatric disorders. The highly tractable mechanism of CYFIP1-FMRP and the regulation of ARC are one such biological pathway, offering a unique entry point for continued study and phenotypic rescue. Future development of novel mechanism-based therapeutic approaches will be vital to meet the ever-growing need to treat these common, yet debilitating, psychiatric disorders.Penetrant risk variants affecting this biological pathway increase psychiatric vulnerability to a range of psychiatric disorders. For example, CNVs affecting The authors have no conflicts of interest to declare.The work was supported by a Wellcome Trust Strategic Award  and a Medical Research Council Centre Grant (GO801418). S.T. and N.E.C. were supported by Cardiff University Neuroscience and Mental Health Research Institute Fellowships. S.T. has been additionally supported by startup funds associated with a new appointment as lecturer at Keele University.The review was conceived by S.T. and written by S.T. and N.E.C.; K.L.T., L.S.W., and J.H. provided additional text, comments, and support. Figures were created by S.T. and N.E.C.


ARHGEF3 and CTNNAL1 map to the RET-dependent HSCR susceptibility loci. We therefore sought to explore whether genetic variants within RET, ARHGEF3 and CTNNAL1, and their genetic interaction networks are associated with HSCR. Taking advantage of a strategy that combined the MassArray system and gene-gene interaction analysis with case-control study, we interrogated 38 polymorphisms within RET, ARHGEF3 and CTNNAL1 in 1015 subjects (502 HSCR cases and 513 controls) of Han Chinese origin. There were statistically significant associations between 20 genetic variants in these three genes and HSCR. Haplotype analysis also revealed some significant global P values, i.e. RET_ rs2435357-rs752978-rs74400468-rs2435353-rs2075913-rs17028-rs2435355 (P = 3.79×10-58). Using the MDR and GeneMANIA platforms, we found strong genetic interactions among RET, ARHGEF3, and CTNNAL1 and our previously studied GAL, GAP43, NRSN1, PTCH1, GABRG2 and RELN genes. These results offer the first indication that genetic markers of RET, ARHGEF3 and CTNNAL1 and relevant genetic interaction networks confer the altered risk to HSCR in the Han Chinese population.Hirschsprung disease (HSCR), the most common enteric neuropathy, stands as a model for complex genetic disorders. It has recently been demonstrated that both  Hirschsprung disease , as a life-threatening genetic disorder, affects approximately 1/5000 live births around the world (2.8/10000 newborns in Asian population), which makes it the most frequent congenital disease of intestinal motility [DENND3, NCLN, NUP98 and TBATA, all of which might play a role in neuronal development [ALDH1A2, PLD1, CASQ2 and CCT2 [The major causes of Hirschsprung disease can be attributed to genetic factors or gene-gene interaction networks, since HSCR presents crucial features of multifactorial genetic models, such as low sex-dependent penetrance, high sibling recurrence risk, high heritability and interfamilial variation . Approxielopment . More reand CCT2 –13.RET , the major gene in HSCR, accounts for more than 80% of all known mutations [RET variants in HSCR [RET mutations also carry modifier loci, which contribute to HSCR presentation and phenotype severity, suggesting the potential interactions between RET and other genes involved in HSCR pathogenesis [Arhgef3 (Rho guanine nucleotide exchange factor (GEF) 3) and Ctnnal1 (catenin (cadherin associated protein), alpha-like 1) have human homologues that map to previously identified HSCR susceptibility loci, i.e. the RET-dependent susceptibility loci (3p21 and 9q31), which makes them excellent candidate genes for Hirschsprung disease [RET, ARHGEF3 and CTNNAL1 contribute to the altered risk of HSCR since the joint gene-gene effects may have a significant impact on HSCR development [RET and PHOX2B polymorphisms substantially affects the risk of Hirschsprung disease, suggesting that HSCR, as a multifactorial genetic disorder, requires the interactions of multiple unlinked genes to produce the phenotype [GAL, GAP43 and NRSN1 contribute to the altered HSCR susceptibility, and importantly, the interaction networks among GAP43, NRSN1 and PTCH1 confer an increased risk to Hirschsprung disease [utations . A recen in HSCR . Moreoveogenesis . Taking  disease –19. In aelopment . It has henotype . In a rey loci 3p and 9q31RET, ARHGEF3 and CTNNAL1 have an impact on the risk of Hirschsprung disease, and by recruiting 1015 subjects and 38 polymorphisms within these three genes, we further explored the potential interaction networks among RET, ARHGEF3, and CTNNAL1 and our previously studied GAL, GAP43, NRSN1, PTCH1, GABRG2 and RELN genes.With all these lines of evidence, we tried to determine whether genetic variants of P> 0.05). The allele and genotype frequencies of the 38 SNPs are listed in RET SNPs, 3 ARHGEF3 SNPs, and 4 CTNNAL1 SNPs. Of note, all 13 positive SNPs in RET survived the FDR correction in terms of both allele and genotype distributions , our present results have shown that the odds ratios have the same magnitudes, suggesting that the cases studied here are representative of Hirschsprung disease  was found in multiple groups of markers, such as RET_rs2435353-rs2075913-rs17028, ARHGEF3_rs11720618-rs11925835-rs3732508 rs3732511-rs1009119-rs6978, CTNNAL1_rs10979650-rs4978766-rs4978379-rs838816-rs7021366 and etc. We therefore investigated the haplotype distributions for these SNPs in the later analysis.We further conducted linkage disequilibrium (LD) analyses of genetic markers within the  studies . Figure D' > 0.7) will markedly increase the statistical power for association with the disease, we then carried out haplotype analyses of SNPs in these three genes. Moreover, haplotypes were omitted from the analysis if the estimated haplotype probabilities were less than 3% in either the HSCR or control group. Only haplotypes with strong LD were selected for presentation , ARHGEF3_rs11720618-rs11925835-rs3732508-rs3772219-rs3732511-rs1009119-rs6978-rs8 08 (P = 2.99×10-8), and CTNNAL1_rs10979650-rs4978 766-rs4978379-rs838816-rs7021366 (P = 5.05×10-5). Importantly, we found that the combinations of the RET risk haplotype reported previously [ARHGEF3 and/or CTNNAL1 showed increased odds ratios compared to those consisting of only the RET risk haplotype and the non-risk alleles of ARHGEF3 and/or CTNNAL1  was the best single factor model  (RET (rs3026737) and ARHGEF3 (rs4681946), and specifically, certain genotype combinations, such as CT (rs3026737)-AA (rs4681946), might lead to an increased risk in HSCR (RET (rs7069590), ARHGEF3 (rs11717604), ARHGEF3 (rs3732508), and CTNNAL1 (rs7021366) constituted the best four-factor model .The multifactor dimensionality reduction (MDR) strategy was then recruited to assess the potential gene-gene interactions among  CTNNAL1 , 2B. Of = 10/10) . Moreove in HSCR . On the RET, ARHGEF3, and CTNNAL1 and our previously studied GAL, GAP43, NRSN1, PTCH1, GABRG2 and RELN genes [RET, ARHGEF3, and CTNNAL1 might be involved in the positive regulation of neuron projection development, regulation of cell projection organization, and small GTPase-mediated signal transduction. These nine genes interacted with each other mainly through co-expression and/or genetic interactions, and some of them might contribute to the same pathways, such as signaling by GPCR.We also used GeneMANIA, a flexible user-friendly database, to explore the functional association networks among LN genes . As showRET, ARHGEF, and CTNNAL1 genes in 502 HSCR cases and 513 normal controls and identified significant associations of these three genes with altered HSCR susceptibility. Moreover, our present work for the first time unraveled that the interaction networks among RET, ARHGEF3, and CTNNAL1 and our previously studied GAL, GAP43, NRSN1, PTCH1, GABRG2 and RELN genes might contribute to an increased risk of HSCR.HSCR is a congenital complex disorder caused by a deficit in the migration process of enteric neural crest cells (ENCCs). To date, more than 15 genes have been linked to the development of Hirschsprung disease, and yet, the landscape of genetic networks regarding HSCR risk has not been fully characterized. Here, we carried out linkage disequilibrium analyses of 38 genetic markers within the RET, which contains at least 80% of all known HSCR-causing mutations [RET were significantly linked to the altered HSCR susceptibility. Of note, certain alleles and/or genotypes of these 13 positive SNPs might be risk factors for HSCR, such as the A allele and AA genotype of rs2505998, the T allele and TT genotype of rs2435357 and the C allele and CC genotype of rs752978, while the others might be protective factors against HSCR, such as the T allele and TT genotype of rs17028 and the C allele and CC genotype of rs2435355. Additionally, the 3 positive markers  may further affect the regulatory mechanisms of gene expression since all of them are located in the 3' UTR region [Recent genetics studies have mainly focused on the major HSCR gene, utations . Our preR region , 26, ourARHGEF3, CTNNAL1 and HSCR, since both of them were indicated as excellent candidate genes for HSCR [ARHGEF3 and CTNNAL1 with Hirschsprung disease. As the positive variant rs3732508 in ARHGEF3 is synonymous, it may not change the amino acid directly, and yet, based on recent studies, synonymous SNPs can affect mRNA splicing, stability and structure as well as protein folding, which may cause changes in protein function and cellular response to therapeutic targets [ARHGEF3 markedly inhibits NPC  cell growth and migration [CTNNAL1, the positive marker, is a missense mutation, which results in a change in the amino acid and thus may substantially have an impact on the function of proteins. CTNNAL1 (α-Catulin) is a cytoplasmic molecule that integrates the crosstalk between nuclear factor-kappa B and Rho signaling pathways, and attenuation of α-Catulin in vitro blocked cell migration and invasion induced by other proteins [ARHGEF3 and CTNNAL1, our work also revealed some potential risk factors for HSCR, such as the C allele and CC genotype of ARHGEF3 rs11720618, the A allele and AA genotype of ARHGEF3 rs3732508, and the G allele and GG genotype of CTNNAL1 rs7021366, all of which might confer an increased risk of HSCR. Importantly, additional replication studies recruiting larger Asian and non-Asian samples and more markers will certainly be needed in the future.We then tried to interrogate the relationship between for HSCR . Our cur targets . In huma targets . A recenigration . On the proteins , 31. RegRET_A-T-C-G-C-T-C-T  and ARHGEF3_G-C-A-A-C-T-G-A , both of which are the potential risk factors for HSCR, whereas CTNNAL1_G-A-C-C-C  exerts a protective effect against HSCR.On the other hand, our current findings also revealed some significant haplotypes, which are relevant to HSCR susceptibility . CompareARHGEF3 and CTNNAL1 map to the RET-dependent HSCR susceptibility loci identified at 3p21 and 9q31, respectively [RET, ARHGEF3 and CTNNAL1 -ARHGEF3 (rs11717604)-ARHGEF3 (rs3732508)-CTNNAL1 (rs7021366), showed the most significant OR compared to other models, further supporting that a multifactor model might play a major role in HSCR susceptibility. Since 2 positive cSNPs (ARHGEF3_rs3732508 and CTNNAL1_rs7021366) were also involved in this best four-factor model, the interaction between them might have a functional impact on HSCR pathogenesis.Interestingly, both ectively , 19. Sinectively , 33. We  CTNNAL1 , 2B, ands7021366 . InteresRET, ARHGEF3, and CTNNAL1, and our previously studied GAL, GAP43, NRSN1, PTCH1, GABRG2 and RELN genes [RET, ARHGEF3 and CTNNAL1 might functionally contribute to the regulation of cell projection organization, small GTPase-mediated signal transduction and positive regulation of neuron projection development, and thus they might be involved in HSCR etiology, as HSCR is due to a deficit in the development of the enteric nervous system.Taking advantage of the GeneMANIA platform, we then explored functionally related gene-gene interaction networks among LN genes . These HRET, ARHGEF3 and CTNNAL1 confer an altered risk to Hirschsprung disease in the Han Chinese population. To the best of our knowledge, this study offers the first indication that the gene-gene interactions among RET, ARHGEF3 and CTNNAL1 contribute to an increased risk of HSCR. Moreover, we have also unraveled the potential interaction networks consisting of 9 HSCR-related genes, which might be involved in HSCR susceptibility. Future research is required to fully understand the complexity of genetic interaction networks involved in HSCR risk and address the genetic basis of Hirschsprung disease.Taken together, our present data show that genetic variants and haplotypes in In the present study, we recruited 1015 subjects, including 502 sporadic HSCR cases  and 513 normal controls . All the participants were of Han Chinese origin and were enrolled from biologically unrelated residents. The 502 cases in the study had the diagnosis of HSCR based on histological examination of surgical or biopsy resection material, including 369 S-HSCR (short segment HSCR), 74 L-HSCR (long segment HSCR) and 59 TCA . Controls were randomly recruited from the general population with no history of chronic constipation. We received approval for the study from Xinhua Hospital and Capital Institute of Pediatrics and obtained written informed consent from participants or their parents after the procedure had been fully explained. DNA was extracted using the QIAamp DNA Blood Midi Kit .http://gvs.gs.washington.edu/GVS150/) with MAF  ≥ 0.15 and r2 ≥ 0.8 according to the HapMap HCB (Han Chinese in Beijing) database. We thus enrolled 38 SNPs in the study, including 34 tagSNPs and 4 SNPs  that have been previously reported [RET: rs17028, rs2742240 and rs2435355; ARHGEF3: rs1009119, rs6978 and rs808), 4 cSNPs (coding SNPs) , and 28 intronic SNPs  analysis was employed to investigate the SNP-SNP interaction networks in regard to Hirschsprung disease. MDR software (version 3.0.2) was thus used to carry out the MDR analysis, and the risk factors were explored in the best model, which maximized both testing accuracy and cross-validation consistency (CVC) . Specifihttp://analysis.bio-x.cn/myAnalysis.php) was used to assess Hardy-Weinberg equilibrium (HWE), odds ratio (OR), 95% confidence interval (CI), allelic and genotypic association, and to study linkage disequilibrium (LD), allelic and haplotype distribution [P values of genetic analysis [P values were two-tailed, and the significance level was set at P = 0.05. G*Power 3 was used to conduct the power calculation [SHEsis (ribution . The falanalysis . All P vculation . Plink sculation .Supplementary Table 1Supplementary Table 2


Ras analog in brain (Rab) proteins are small guanosine triphosphatases (GTPases) that belong to the Ras-like GTPase superfamily, and they can regulate vesicle trafficking. Rab proteins alternate between an activated (GTP-bound) state and an inactivated (GDP-bound) state. Early endosome marker Rab5 GTPase, a key member of the Rab family, plays a crucial role in endocytosis and membrane transport. The activated-state Rab5 recruits its effectors and regulates the internalization and trafficking of membrane receptors by regulating vesicle fusion and receptor sorting in the early endosomes. In this review, we summarize the role of small Rab GTPases Rab5 in membrane receptor trafficking and the activation of signaling pathways, such as Ras/MAPK and PI3K/Akt, which ultimately affect cell growth, apoptosis, tumorigenesis, and tumor development. This review may provide some insights for our future research and novel therapeutic targets for diseases. Ras analog in brain (Rab) proteins, belonging to the largest family of Ras superfamily, are small guanosine diphosphate (GTP)- bound proteins that regulate intracellular trafficking pathways . There aRab5 is one of the most crucial members of the Rab family, whose functions and mechanisms are well studied. Rab5 transforms between the activated form, GTP-bound Rab5 (GTP-Rab5), and the inactivated form, guanosine diphosphate (GDP)- bound Rab5 (GDP-Rab5) . ActivatIn this review, we discussed the structure and activation of Rab5 and highlighted the recent advancements in the Rab5 regulating membrane receptor trafficking and signaling pathways, which will finally affect the occurrence and development of diseases.S. cerevisiae by Novick. It was found a series of genes are necessary for the yeast secretion, which were named SEC  [YPT1 protein in yeast S. cerevisiae [The Rab GTPase proteins were first studied in yeast 2, etc.) . Subsequrevisiae . Furtherrevisiae .β sheets (β1–β6), five α helixes (α1–α5), and five polypeptide rings; N- and C-terminals; and the molecular switches I and II [Rab proteins share similar structures, generally containing two cysteine residues at the carboxyl terminus generally, which appear in the form of -CC, -CXC, -CCXX, -CXXX, or -CCXXX (X represents any amino acid) and act as the membrane localization signal . The keyI and II . The N-tRab GTPases can transform between the GTP-bound activated form and GDP-bound inactivated form . The GTPβ sheets and α helixes are folded at the N terminus, and -CCXX structure and p-loop structure are at the C-terminal. The -CCXX structure is often modified by prenylation, contributing to the location of Rab5 in the plasma membrane. P-loop consists of three parts: (1) 27–34 residues induce the hydrolysis, binding, and dissociation of GTP in Rab5, (2) 49–51 residues act as switch I, and (3) 79–81 residues act as switch II [Rab5 is a key member of the Rab family, and Rab5A is its most important subtype, with well-identified functions and mechanisms. Rab5 is mentioned as Rab5A in most studies. Rab5A is located at 3p24.3 and is composed of 215 amino acids with a molecular weight of 23.658 kD . The prowitch II .The present studies on the mutants of Rab5 focus on S34N, Q79L, A30P, G78I, N125I, N133I, D136N, and C-terminal and N-terminal truncations. Wherein, Rab5-S34N, a persistently inactive form of Rab5, is a guanylate-bound deficient mutant, and preferable to bind GDP. Overexpression of the dominant negative Rab5-S34N inhibits fusion of early endosomes and endocytosis of transferring . Rab5-Q7Rab5 transforms between the activated form GTP-Rab5 and inactivated form of GDP-Rab5. The activation of Rab5 is regulated by GEFs, and the inactivation is regulated by GAPs . GEFs coRab5 recruits the effector proteins via their GTP-dependent switch I and II to distinct subcellular compartments to regulate membrane trafficking events. The crucial effectors of Rab5 are early endosome antigen-1 (EEA1) , 29, rab2H2 zinc finger structure and C-terminal domain [EEA1, a key effector of Rab5 with a molecular weight of 162 kD, is a biomarker for early endosomes and has a parallel coiled-coil homodimer structure. EEA1 contains two binding sites for Rab5 : N-terminal Cl domain , which cl domain . Then, Rl domain  and fusel domain .Rabaptin-5 is another Rab5 effector that plays a crucial role in membrane docking . Rab5 inThere is close interaction among GEFs, Rab5, and Rab5 effectors. For example, after activation by Rabex-5, Rab5 recruits its effector Vps15 to interact with phosphatidylinositol 3-kinase (PI3K). Then, PI3K generates phosphatidylinositol 3-phosphate, which further recruit more effectors to interact with Rab5. Moreover, activated Rab5 interacts with its effector rabaptin-5 to form a complex. Rabaptin-5 further promotes the activity of Rabex-5 to facilitate the positive feedback from GTP-Rab5 and the binding of Rab5 to its downstream effectors .Rab5 affects the internalization and intracellular transport of receptors, such as receptor tyrosine kinases (RTKs), G protein-coupled receptors (GPCRs), and antigen recognition receptors by recruiting Rab5 effectors. The signal transduction of receptors occurs in early endosomes, further affecting gene transcription and ultimately affecting cell morphology, growth, differentiation, apoptosis, and disease development as shown in RTKs are a large superfamily of receptors that can bind with ligands and phosphorylate tyrosine residues of the target proteins through tyrosine kinase domains. They have similar structures, including the extracellular glycosylated peptides, which are responsible for binding to ligands, hydrophobic transmembrane domain, and the intracellular region with tyrosine kinase activity , 45. RTKEndocytosis of RTKs includes internalization, transport, sorting, and degradation , 49, whiα [The EGF receptor is the most widely studied molecules among RTKs. The extracellular region of the EGF receptor consists of 622 amino acid residues, which bind multiple kinds of ligands including EGF and TGFα . In addiα . Knockdoα .CMTM3, a tumor suppressor gene, decreased EGF receptor expression and EGF-mediated tumorigenicity by promoting Rab5 activity in gastric cancer [GEFs or interacting proteins of Rab5, such as phospholipase D (PLD), hypoxia inducible factor (HIF), neuropilin-2 (NRP2)/WDFY1 axis, and leucine-rich repeat kinase 2 (LRRK2), may also regulate the internalization, transport, and downstream signaling pathways of the EGF receptor . In addic cancer .PLD can directly affect the upstream molecules of the EGF receptor and interact with GAP proteins, during which, Rab5 regulates EGF receptor endocytosis, clathrin vesicles formation, and finally affects EGF receptor function . By down C-MET is the receptor for HGF, which is involved in cell proliferation, differentiation, and signal transduction and regulation of cytoskeleton rearrangement. C-MET is closely associated with tumorigenesis and development of various cancers. Rab5 is also involved in the transport and signal transduction of c-MET. PTP1B interacts with c-MET, EGF, and PDGF receptors, affecting their internalization. Deletion of PTP1B promotes the phosphorylation of NSF and reduces the formation of phosphatidylinositol 3-phosphate-positive early endosomes and the activation of Rab5, resulting in inhibition of c-MET and EGF receptor transport and degradation . KnockdoIn addition, Rab5 plays a role in PDGF receptor internalization and trafficking . P85, a δ  and inhibits colony-stimulating factor 1 receptor downstream Akt signaling pathway, ultimately affecting the function of macrophages [Rab5 affects the internalization, trafficking, and signal transduction of the VEGF receptor and colony-stimulating factor 1 receptor. Overexpression of Rab5-Q79L in endothelial cells increases the size of early endosomes and induces the colocalization of EEA1 and VEGF receptors in endosomes, while knockdown of Rab5 enhances the activation of VEGF receptor (Y1175)/MAPK p42/44 signaling pathway . Colony-rophages .GPCR family, the largest and the most important membrane receptor superfamily in human, has more than 2000 members and is involved in virtually all life activities. The structure of GPCRs includes extracellular N-terminal domain, seven transmembrane helices (TM1-TM7) , intraceβ-arrestins to (1) inhibit the interaction of GPC receptors with G proteins, resulting in signal termination and (2) promoting endocytosis of GPC receptors, most of which are mediated by clathrin and catalyzed by dynamin [β-Arrestin induces the trafficking of GPCRs to the coated pits via β2-adaptin and clathrin [Upon ligands stimulation, GPCRs are phosphorylated rapidly by GPCR kinases, and they bind to adapter protein  dynamin , 73. Theclathrin . After iclathrin , 76.Rab5 is involved in the internalization and trafficking of GPCRs by regulating vesicle fusion and receptor sorting in early endosomes . The traβ-catenin signaling pathway and affects disease development [Lysophosphatidic acid (LPA) is involved in metabolism, signal transduction, regulation of organ function, and is associated with inflammation , 81 and elopment , 91.In addition, Rab5 colocalizes with the oxytocin receptor , CXCR2 , and othIn addition to the aforementioned receptors, Rab5 is involved in the transport and signal transduction of antigen recognition receptors, such as pattern-recognition receptors (PRRs) in innate immune cells, T-cell receptor (TCR), and B-cell receptor (BCR) in adaptive immune cells.PRRs can recognize pathogen-associated molecular patterns, which can activate a series of signaling pathways and trigger innate immune responses. PRRs include toll-like receptors (TLRs), C-type lectin receptors, NOD-like receptors, RIG-I-like receptor, and DNA-sensing molecules in the cytoplasm .Hif-1 and CCL2, ultimately promoting the amplification of bone marrow-derived multifunctional hematopoietic stem cells [By binding with TLR4, lipopolysaccharide activates inflammatory-related cells and leads to inflammation . The colem cells .Mannose and scavenger receptors are macrophage surface receptors, which participate in pathogen recognition, antigens presentation, and maintain homeostasis , 99. Bot+CD8+ thymocytes is obviously reduced in T-cell-specific Rab5-N133I transgenic mice, suggesting that Rab5 plays a key role in TCR transport and signal transduction [In addition, Rab5 is involved in the transport and signal transduction of TCR and BCR. TCR forms complex with Rab5 in early endosomes and accumulates in Rab5-positive early endosomes . The redsduction .The internalization of BCR and BCR-mediated signal transduction establish a series of checkpoints in B cells to ensure B-cell maturation, BCR receptor formation, and humoral immune response generation . Upon anOverall, there are two methods we suggest to treat diseases according to the current Rab5-related studies. First, a direct interaction with Rab5, such as Rab5-targeted therapies, that can transform the activation of Rab5 and influence receptor internalization and trafficking, leading to physiological changes of patients. Second, an indirect way to regulate Rab5 by influencing Rab5 effectors, GEFs, or GAPs is another potential strategy via influencing the aberrant expression, internalization, trafficking, and degradation of receptors. At present, the clinical cancer treatment, for instance, is difficult to achieve satisfactory prognosis of cancer patients because of the high recurrence and metastasis tendency after surgery, and the resistance to radiotherapy and chemotherapy. Thus, regulation of the Rab5 strategy may relieve the cancer patient's distress and provide us a novel idea for cancer therapy.Rab5 is a key factor in regulating early endocytosis. Rab5, recruits its effectors to early endosomes, is involved in the transport of endosomes, and affects membrane receptor internalization, trafficking, and related signaling pathways, which contribute to gene transcription and the biological processes of cells. The mutation of Rab5 can cause abnormal cell morphology and function, suggesting that the structure of Rab5 is closely related to its function and occurrence and development of diseases. However, the mechanisms of Rab5 in diseases are not fully understood and need further investigation.In summary, the study of Rab5 will help us understand the regulation mechanisms of receptor internalization and trafficking and provide new ideas and targets for the treatment of related diseases.