The massive use of face masks during and after the COVID-19 pandemic has raised global concerns about environmental issues. Microplastics released from face masks pose great threats to ecosystems and human health. However, the potentially hazardous effects of face mask-derived microplastics (FMMs) on humans remain poorly understood. Using neural organoid models aims to reveal the toxicity of FMMs to human early neural development. Retinal organoids derived from human embryonic stem cells were exposed to FMMs for 21 days during early retinogenesis. FMMs were internalized by retinal organoids. Exposure to FMMs disrupted the growth and development of retinal organoids in dose- and time-dependent manners, as evidenced by abnormal morphologies. Aberrant cell events, such as cell proliferation, apoptosis, and differentiation contributed to the disarrangement of the neural retina. Transcriptome data proved that the neurotoxicity of FMMs was closely related to disordered neurogenesis, anatomical structure morphogenesis, and axon guidance. Co-exposure to triphenyl phosphate (a common organophosphate flame retardant) and FMMs exhibited more pronounced neurotoxicity than FMM exposure alone. These findings are expected to uncover the potential threats of FMMs to human neurodevelopment and emphasize the importance of optimizing the management and safe disposal of used face masks.

During cardiac development, the heart is innervated by the autonomous nervous system. After development, neurons of the autonomic nervous system have limited capacity for growth and regeneration. However, in recent decades, it has become clear that cardiac nerves can regenerate after cardiac damage. Excessive reinnervation, so-called sympathetic hyperinnervation, may render patients vulnerable to ventricular arrhythmias and heart failure. Several studies have investigated axonal guidance cues as mediators of cardiac innervation. Axonal guidance cues direct neuronal growth of the axon and play a significant role in the regeneration and remodelling of cardiac autonomic innervation after cardiac damage. This review focusses on the current literature regarding the axonal guidance cue group of semaphorins and their function in the healthy and diseased postnatal heart. In view of cardiac innervation, most studies have focussed on semaphorin 3A (SEMA3A), whereas less is known about the function of the other semaphorin classes. SEMA3A is a neuronal repellent and is associated with a decrease in the density of sympathetic neurons in the heart. Its decline in expression after myocardial infarction plays a role in the development of sympathetic hyperinnervation and the subsequent increased risk of ventricular arrhythmias. In congestive heart failure, the opposite occurs: an increase in SEMA3A expression underlies decreased nerve density that may also serve as a substrate for ventricular arrhythmias. Although the literature on their role in cardiac innervation is still relatively scarce, semaphorins, especially SEMA3A, seem worthwhile to consider when exploring options to modulate pathologic innervation patterns in cardiovascular disease.

Aging serves as a pivotal factor in the etiology of numerous diseases, such as Alzheimer's disease (AD), Parkinson's disease, diabetes, osteoarthritis, atherosclerosis and aging-related macular degeneration. Notably, these diseases often interact with AD through various pathways, facilitating the onset or progression of one another. Semaphorin 3 A (Sema3A), a protein that is essential for axonal guidance during neural development, has recently been identified as a novel regulator in the pathogenesis and progression of multiple aging-related diseases. This article provides a comprehensive review of the expression patterns and mechanisms of action of Sema3A in these diseases. Specifically, Sema3A influences the occurrence and development of aging-related diseases by participating in oxidative stress, inflammatory responses, apoptosis, and synaptic plasticity. Therefore, therapeutic strategies targeting Sema3A present promising avenues for delaying the progression of aging-related diseases and offer novel insights and strategies for their treatment.

Somatic stem cell pools comprise diverse, highly specialized subsets whose individual contribution is critical for the overall regenerative function. In the bone marrow, myeloid-biased hematopoietic stem cells (myHSCs) are indispensable for replenishment of myeloid cells and platelets during inflammatory response but, at the same time, become irreversibly damaged during inflammation and aging. Here we identify an extrinsic factor, Semaphorin 4A (Sema4A), which non-cell-autonomously confers myHSC resilience to inflammatory stress. We show that, in the absence of Sema4A, myHSC inflammatory hyper-responsiveness in young mice drives excessive myHSC expansion, myeloid bias and profound loss of regenerative function with age. Mechanistically, Sema4A is mainly produced by neutrophils, signals via a cell surface receptor, Plexin D1, and safeguards the myHSC epigenetic state. Our study shows that, by selectively protecting a distinct stem cell subset, an extrinsic factor preserves functional diversity of somatic stem cell pool throughout organismal lifespan.

Oligodendrocyte progenitor cells (OPCs) are highly dynamic, widely distributed glial cells of the central nervous system responsible for generating myelinating oligodendrocytes throughout life. However, the rates of OPC proliferation and differentiation decline dramatically with aging, which may impair homeostasis, remyelination and adaptive myelination during learning. To determine how aging influences OPCs, we generated a transgenic mouse line (Matn4-mEGFP) and performed single-cell RNA sequencing, providing enhanced resolution of transcriptional changes during key transitions from quiescence to proliferation and differentiation across the lifespan. We found that aging induces distinct transcriptomic changes in OPCs in different states, including enhanced activation of HIF-1α and WNT pathways. Pharmacological inhibition of these pathways in aged OPCs was sufficient to increase their ability to differentiate in vitro. Ultimately, Matn4-mEGFP mouse line and the sequencing dataset of cortical OPCs across ages will help to define the molecular changes guiding OPC behavior in various physiological and pathological contexts.

Helicobacter pylori (H. pylori) infection is one of the major risk factors of stomach cancer. Strains carrying the oncogenic cytotoxin CagA (cytotoxin-associated gene A) induce epithelial-mesenchymal transition (EMT) and contribute to tumor progression and metastasis. However, the mechanism in which CagA induces EMT has not been defined. In this study, using genetic methods in Drosophila, we identified Semaphorin 5A (SEMA5A) as a new target for CagA. We showed that infection with CagA-positive H. pylori downregulated the expression level of SEMA5A to induce expression of EMT-driving transcription factor Snail and mesenchymal marker N-cadherin, and promote invasive behavior in gastric epithelial cells. Furthermore, we demonstrated that transient over-expression of SEMA5A in H. pylori-infected cells inhibited CagA-mediated gain of mesenchymal phenotype. These results suggest that SEMA5A could be a key mediator of EMT and gastric carcinogenesis caused by CagA-positive H. pylori infection.

Background: Meniere's disease (MD) is a set of rare disorders that affects >4 million people worldwide. Individuals with MD suffer from episodes of vertigo associated with fluctuating sensorineural hearing loss and tinnitus. Hearing loss can involve one or both ears. Over 10% of the reported cases are observed in families, suggesting its significant genetic contribution. The condition is polygenic with >20 genes, and several patterns of inheritance have been reported, including autosomal dominant, autosomal recessive, and digenic inheritance across multiple MD families. Preclinical research using animal models has been an indispensable tool for studying the neurophysiology of the auditory and vestibular systems and to get a better understanding of the functional role of genes that are involved in the hearing and vestibular dysfunction. While mouse models are the most used preclinical model, this review analyzes alternative animal and non-animal models that can be used to study MD genes. Methods: A literature search of the 21 genes reported for familial MD and the preclinical models used to investigate their functional role was performed. Results: Comparing the homology of proteins encoded by these genes to other model organisms revealed Drosophila and zebrafish as cost-effective models to screen multiple genes and study the pathophysiology of MD. Conclusions: Murine models are preferred for a quantitative neurophysiological assessment of hearing and vestibular functions to develop drug or gene therapy.

Background/Objectives: The experience of prenatal stress results in various physiological disorders due to an alteration of an offspring's methylome and transcriptome. The objective of this study was to determine whether PNS affects DNA methylation (DNAm) and gene expression in the stress axis tissues of mature Brahman cows. Methods: Samples were collected from the paraventricular nucleus (PVN), anterior pituitary (PIT), and adrenal cortex (AC) of 5-year-old Brahman cows that were prenatally exposed to either transportation stress (PNS, n = 6) or were not transported (Control, n = 8). The isolated DNA and RNA samples were, respectively, used for methylation and RNA-Seq analyses. A gene ontology and KEGG pathway enrichment analysis of each data set within each sample tissue was conducted with the DAVID Functional Annotation Tool. Results: The DNAm analysis revealed 3, 64, and 99 hypomethylated and 2, 93, and 90 hypermethylated CpG sites (FDR < 0.15) within the PVN, PIT, and AC, respectively. The RNA-Seq analysis revealed 6, 25, and 5 differentially expressed genes (FDR < 0.15) in the PVN, PIT, and AC, respectively, that were up-regulated in the PNS group relative to the Control group, as well as 24 genes in the PIT that were down-regulated. Based on the enrichment analysis, several developmental and cellular processes, such as maintenance of the actin cytoskeleton, cell motility, signal transduction, neurodevelopment, and synaptic function, were potentially modulated. Conclusions: The methylome and transcriptome were altered in the stress axis tissues of mature cows that had been exposed to prenatal transportation stress. These findings are relevant to understanding how prenatal experiences may affect postnatal neurological functions.

Development of neuronal connections is spatially and temporally controlled by extracellular cues which often activate their cognate cell surface receptors and elicit localized cellular responses. Here, we demonstrate the use of an optogenetic tool to activate receptor signaling locally to induce actin-mediated growth cone remodeling in neurons. Based on the light-induced interaction between Cryptochrome 2 (CRY2) and CIB1, we generated a bicistronic vector to co-expresses CRY2 fused to the intracellular domain of a guidance receptor and a membrane-anchored CIB1. When expressed in primary neurons, activation of the growth inhibitory PlexA4 receptor induced growth cone collapse, while activation of the growth stimulating TrkA receptor increased growth cone size. Moreover, local activation of either receptor not only elicited the predicted response in light-activated growth cones but also an opposite response in neighboring no-light-exposed growth cones of the same neuron. Finally, this tool was used to reorient growth cones toward or away from the site of light activation and to stimulate local actin polymerization for branch initiation along axonal shafts. These studies demonstrate the use of an optogenetic tool for precise spatial and temporal control of receptor signaling in neurons and support its future application in investigating cellular mechanisms of neuronal development and plasticity.

A substantial portion of patients infected with SARS-CoV-2 experience prolonged complications, known as Long COVID (LC). A subset of these patients exhibits the most debilitating symptoms, similar to those defined in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). We performed bulk RNA sequencing (RNAseq) on the whole blood of LC with ME/CFS, at least 12 months post-onset of the acute disease, and compared them with controls. We found that LC patients had a distinct transcriptional profile compared to controls. Key findings include the upregulation of genes involved in immune dysregulation and neuronal development, such as Fezf2, BRINP2, HOXC12, MEIS2, ZFHX3, and RELN. These genes are linked to neuroinflammatory responses, cognitive impairments, and hematopoietic disturbances, suggesting ongoing neurological and immune disturbances in LC patients. RELN, encoding the Reelin protein, was notably elevated in LC patients, potentially serving as a biomarker for LC pathogenesis due to its role in inflammation and neuronal function. Immune cell analysis showed altered profiles in LC patients, with increased activated memory CD4 + T cells and neutrophils, and decreased regulatory T cells and NK cells, reflecting immune dysregulation. Changes in cytokine and chemokine expression further underscore the chronic inflammatory state in LC patients. Notably, a unique upregulation of olfactory receptors (ORs) suggest alternative roles for ORs in non-olfactory tissues. Pathway analysis revealed upregulation in ribosomal RNA processing, amino acid metabolism, protein synthesis, cell proliferation, DNA repair, and mitochondrial pathways, indicating heightened metabolic and immune demands. Conversely, downregulated pathways, such as VEGF signaling and TP53 activity, point to impaired tissue repair and cellular stress responses. Overall, our study underscores the complex interplay between immune and neuronal dysfunction in LC patients, providing insights into potential diagnostic biomarkers and therapeutic targets. Future research is needed to fully understand the roles and interactions of these genes in LC pathophysiology.

Lymphatic vessels and blood vessels have some similarities in structure, but they have distinct contraction characteristics and functions. Revealing the detailed transcriptional differences of lymphatic, artery and vein are required for circulation research.

The tissues of the mesenteric lymphatic, artery, and vein were collected from Wistar rats. The transcriptome signatures of these tissues from RNA-seq (RNA sequencing) were analyzed using bioinformatic methods.

GO (gene ontology) enrichment showed the three tissues have distinct gene expression patterns in extracellular matrix, cell adhesion molecule binding, receptor ligand activity, and contractile fiber. The genes involved in cell contractility were also differently expressed, which were enriched into the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways of cytoskeleton in muscle cells, vascular smooth muscle contraction, and renin-angiotensin system. Through PPI (protein-protein interaction) analysis, we identified 43 differently expressed hub genes in the three tissues. Thirty-four transcription factors and cofactors were identified as important for the normal function of the three tissues. Furthermore, we screened out 20 potential marker genes for each tissue.

Our study described the transcriptome signatures of mesenteric lymphatic, artery, and vein, shedding light on the distinct contraction mechanisms of these tissues. These results also provided potential therapeutic targets for circulation diseases and potential markers for lymphatic and blood vessel studies.

The Australian brushtail possum (Trichosurus vulpecula) is adapted to a wide range of food plants across its range and is exposed to numerous physiological challenges. Populations that are resistant to the plant toxin sodium fluoroacetate are of particular interest as this compound has been used since the 1940s for vertebrate pest management around the world. Candidate gene identification is an important first step in understanding how spatial populations have responded to local selection resulting in local physiological divergence. We employ differential gene expression of liver samples from wild-caught brushtail possums from toxin-resistant and toxin-susceptible populations to identify candidate genes that might be involved in metabolic pathways associated with toxin-resistance. This allowed us to identify genetic pathways involved in resistance to the plant toxin sodium fluoroacetate in Western Australian possums but not those originally from south eastern Australia. We identified differentially expressed genes in the liver that are associated with cell signalling, encapsulating structure, cell mobility, and tricarboxylic acid cycle. The gene expression differences detected indicate which metabolic pathways are most likely to be associated with sodium fluoroacetate resistance in these marsupials and we provide a comprehensive list of candidate genes and pathways to focus on for future studies.

Plexins are a family of transmembrane receptors known for their diverse roles in neural development, axon guidance, neuronal migration, synaptogenesis, and circuit formation. Semaphorins are a class of secreted and membrane proteins that act as primary ligands for plexin receptors. Semaphorins play a crucial role in central nervous system (CNS) development by regulating processes such as axonal growth, neuronal positioning, and synaptic connectivity. Various types of semaphorins like sema3A, sema4A, sema4C, sema4D, and many more have a crucial role in developing brain diseases. Likewise, various evidence suggests that plexin receptors are of four types: plexin A, plexin B, plexin C, and plexin D. Plexins have emerged as crucial regulators of neurogenesis and neuronal development and connectivity. When bound to semaphorins, these receptors trigger two major networking cascades, namely Rho and Ras GTPase networks. Dysregulation of plexin networking has been implicated in a myriad of brain disorders, including autism spectrum disorder (ASD), Schizophrenia, Alzheimer's disease (AD), Parkinson's disease (PD), and many more. This review synthesizes findings from molecular, cellular, and animal model studies to elucidate the mechanisms by which plexins contribute to the pathogenesis of various brain diseases.

The central nervous system (CNS) is wired by a complex network of integrated glial and neuronal signals, which is critical for its development and homeostasis. In this context, glia-glia communication is a complex and dynamic process that is essential for ensuring optimal CNS function. Semaphorins, which include secreted and transmembrane molecules, and their receptors, mainly found in the plexin and neuropilin families, are expressed in a wide range of cell types, including glia. In the CNS, semaphorin signalling is involved in a spectrum of processes, including neurogenesis, neuronal migration and wiring, and glial cell recruitment. Recently, semaphorins and plexins have attracted intense research aimed at elucidating their roles in instructing glial cell behavior during development or in response to inflammatory stimuli. In this review, we provide an overview of the multifaceted role of semaphorins in glia-glia communication, highlighting recent discoveries about semaphoring-dependent regulation of glia functions in healthy conditions. We also discuss the mechanisms of gliaglia crosstalk mediated by semaphorins under pathological conditions, and how these interactions may provide potential avenues for therapeutic intervention in neuroinflammation-mediated neurodegeneration.

Astrocytes exhibit diverse cellular and molecular properties across the central nervous system (CNS). Recent studies identified region-specific transcription factors (TF) that oversee these diverse properties; how sex differences intersect with region-specific transcriptional programs to regulate astrocyte function is unknown. Here, we show that the TF Nkx6.1 is specifically expressed in ventral astrocytes of the spinal cord and that its deletion results in sex-specific effects on astrocyte morphology. Astrocytes from males exhibit enhanced morphological complexity, accompanied by increased motor function and cholinergic synapses. In contrast, female astrocytes exhibit reduced complexity and no changes in motor function. Mechanistically, we found that Nkx6.1 exhibits sex-specific DNA-binding properties and epigenomic remodeling, identifying Semaphorin 4A (Sema4A) and Gabbr1 as targets regulating astrocyte morphology and cholinergic synapse formation. Collectively, our studies identify astrocytic Nkx6.1 as a key regulator of astrocyte properties in the spinal cord while adding sexual dimorphism as a layer of transcriptional regulation to astrocyte function and circuit activity.

Astrocytoma is a common type of glioma and a frequent cause of brain tumour-related epilepsy. Although the link between glioma and epilepsy is well established, the precise mechanisms underlying epileptogenesis in astrocytoma remain poorly understood. In this study, we performed proteomic analysis of astrocytoma tissue from patients with and without seizures using mass spectrometry-based techniques. We detected 131 differentially expressed proteins (42 upregulated and 89 downregulated). Proteins upregulated in patients with seizures were mostly related to an increase in energy metabolism. Moreover, glial fibrillary acidic protein, which is involved in maintaining normal axonal structures, was abnormally highly expressed in patients with seizures. Proteins downregulated in patients with seizures included those involved in trans-synaptic signalling and gamma-aminobutyric acid synaptic transmission. Interestingly, comparison of protein expression profiles from our cohort with those from a previous study of patients with epilepsy due to other causes showed that the collapsin response mediator protein family of axonal growth regulators was highly expressed only in patients with seizures due to astrocytomas. Further studies of the proteins identified here are required to determine their potential as biomarkers and therapeutic targets.

Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy requires therapeutic combinations that induce quality T cells. Tumor microenvironment (TME) analysis following therapeutic interventions can identify response mechanisms, informing design of effective combinations. We provide a reference single-cell dataset from tumor-infiltrating leukocytes (TILs) from a human neoadjuvant clinical trial comparing the granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting allogeneic PDAC vaccine GVAX alone, in combination with anti-PD1 or with both anti-PD1 and CD137 agonist. Treatment with GVAX and anti-PD-1 led to increased CD8+ T cell activation and expression of cytoskeletal and extracellular matrix (ECM)-interacting components. Addition of CD137 agonist increased abundance of clonally expanded CD8+ T cells and increased immunosuppressive TREM2 signaling in tumor associated macrophages (TAMs), identified by comparison of ligand-receptor networks, corresponding to changes in metabolism and ECM interactions. These findings associate therapy with GVAX, anti-PD1, and CD137 agonist with enhanced CD8+ T cell function while inducing alternative immunosuppressive pathways in patients with PDAC.

Amyloid-β peptide (Aβ) is a critical cause of Alzheimer's disease (AD). It is generated from amyloid precursor protein (APP) through cleavages by β-secretase and γ-secretase. γ-Secretase, which includes presenilin, is regulated by several stimuli. Tau protein has also been identified as a significant factor in AD. In particular, Tau phosphorylation is crucial for neuronal impairment, as phosphorylated Tau detaches from microtubules, leading to the formation of neurofibrillary tangles and the destabilization of the microtubule structure. This instability in microtubules damages axons and dendrites, resulting in neuronal impairment. Notably, Aβ is linked to Tau phosphorylation. Another crucial factor in AD is neuroinflammation, primarily occurring in the microglia. Microglia possess several receptors that bind with Aβ, triggering the expression and release of an inflammatory factor, although their main physiological function is to phagocytose debris and pathogens in the brain. NF-κB activation plays a major role in neuroinflammation. Additionally, the production of reactive oxygen species (ROS) in the microglia contributes to this neuroinflammation. In microglia, superoxide is produced through NADPH oxidase, specifically NOX2. Rho GTPases play an essential role in regulating various cellular processes, including cytoskeletal rearrangement, morphology changes, migration, and transcription. The typical function of Rho GTPases involves regulating actin filament formation. Neurons, with their complex processes and synapse connections, rely on cytoskeletal dynamics for structural support. Other brain cells, such as astrocytes, microglia, and oligodendrocytes, also depend on specific cytoskeletal structures to maintain their unique cellular architectures. Thus, the aberrant regulation of Rho GTPases activity can disrupt actin filaments, leading to altered cell morphology, including changes in neuronal processes and synapses, and potentially contributing to brain diseases such as AD.

The thymus is a primary lymphoid organ composed of a three-dimensional (3D) epithelial network that provides a specialized microenvironment for the phenotypical and functional maturation of lymphoid progenitors. The specification of the pharyngeal endoderm to thymus fate occurs during the early stages of thymic organogenesis, independent of the expression of the transcription factor Foxn1. However, Foxn1 governs the later organogenesis of thymus together with the colonizing lymphoid cells. In the present chapter, we will review recent evidence on the topic covered in our original chapter (Muñoz and Zapata 2019). It described the early development of thymus and its resemblance to the development of endoderm-derived epithelial organs based on tubulogenesis and branching morphogenesis as well as the molecules known to be involved in these processes.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Extracellular vesicles are lipid-bilayer encapsulated nanovesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived extracellular vesicles (KDEVs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEVs in a well-established high-fat diet mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEVs through size-exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of high-fat diet KDEVs compared to KDEVs obtained from control mice on a regular diet, including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEVs are retrogradely trafficked into the dorsal root ganglion (DRG) neuron cell bodies. This study presents the first comprehensive isolation and molecular characterization of the KDEV protein and microRNA cargo in RD and HFD mice. Our findings suggest a potential novel communication pathway between keratinocytes and DRG neurons in the skin, which could have implications for PDN.

Semaphorins and their receptors plexins are axon guidance molecules that navigate axons to their final destinations during neural development. Semaphorins and plexins exert distinct roles in regulating biological functions such as the immune system and bone homeostasis. They also participate in the development and progression of various diseases such as osteoporosis and allergic diseases. This review describes the varied phenotypes revealed by the analysis of semaphorin or plexin knockout mice and discusses the association with pathogenesis and therapy of atherosclerosis, agenesis of the corpus callosum, and neuropsychiatric diseases. The deletion of semaphorin 4D in atherosclerosis-prone Apolipoprotein E-deficient mice mitigated atherosclerotic lesions, indicating its crucial involvement in the progression of atherosclerosis. Semaphorin 4D is also implicated in apoptosis induced by the estrogen-dependent generation of soluble semaphorin 4D and the active form of plexin-B1 in the postnatal vaginal opening in mice. Plexin-A1 knockout BALB/cA mice exhibited the agenesis of corpus callosum. This study indicates the crucial role of plexin-A1 in the midline crossing of callosal pioneer axons projecting from the cerebral cortex during the early phase of callosal formation. Adult plexin-A1-deficient mice exhibit reduced prepulse inhibition deficit, an endophenotype of schizophrenia, in addition to excessive self-grooming. Parvalbumin-expressing interneurons in the medial prefrontal cortex are significantly decreased in plexin-A1 knockout mice. In the parvalbumin neurons, oxidative stress is significantly increased in plexin-A1 knockout mice. Accordingly, plexin-A1 deficiency may augment oxidative stress in parvalbumin neurons, thereby impairing the parvalbumin neuron network and leading to behavioral abnormalities relevant to neuropsychiatric diseases.

Axon pathfinding is a highly dynamic process regulated by the interactions between cell-surface guidance receptors and guidance cues present in the extracellular environment. During development, precise axon pathfinding is crucial for the formation of functional neural circuits. The spatiotemporal expression of axon guidance receptors helps the navigating axon make correct decisions in a complex environment comprising both attractive and repulsive guidance cues. Axon guidance receptors initiate distinct signaling cascades that eventually influence the cytoskeleton at the growing tip of an axon, called the growth cone. The actin cytoskeleton is the primary target of these guidance signals and plays a key role in growth cone motility, exploration, and behavior. Of the many regulatory molecules that modulate the actin cytoskeleton in response to distinct guidance signals, Rho GTPases play central roles. Rho GTPases are molecular switchboards; their ON (GTP-bound) and OFF (GDP-bound) switches are controlled by their interactions with proteins that regulate the exchange of GDP for GTP or with the proteins that promote GTP hydrolysis. Various upstream signals, including axon guidance signals, regulate the activity of these Rho GTPase switch regulators. As cycling molecular switches, Rho GTPases interact with and control the activities of downstream effectors, which directly influence actin reorganization in a context-dependent manner. A deeper exploration of the spatiotemporal dynamics of Rho GTPase signaling and the molecular basis of their involvement in regulating growth cone actin cytoskeleton can unlock promising therapeutic strategies for neurodevelopmental disorders linked to dysregulated Rho GTPase signaling. This review not only provides a comprehensive overview of the field but also highlights recent discoveries that have considerably advanced our understanding of the complex regulatory roles of Rho GTPases in modulating actin cytoskeleton arrangement at the growth cone during axon guidance.

The notochord is an axial structure required for the development of all chordate embryos, from sea squirts to humans. Over the course of more than half a billion years of chordate evolution, in addition to its structural function, the notochord has acquired increasingly relevant patterning roles for its surrounding tissues. This process has involved the co-option of signaling pathways and the acquisition of novel molecular mechanisms responsible for the precise timing and modalities of their deployment. To reconstruct this evolutionary route, we surveyed the expression of signaling molecules in the notochord of the tunicate Ciona, an experimentally amenable and informative chordate. We found that several genes encoding for candidate components of diverse signaling pathways are expressed during notochord development, and in some instances, display distinctive regionalized and/or lineage-specific patterns. We identified and deconstructed notochord enhancers associated with TGF-β and Ctgf, two evolutionarily conserved signaling genes that are expressed dishomogeneously in the Ciona notochord, and shed light on the cis-regulatory origins of their peculiar expression patterns.

Extracellular vesicles are easily accessible in various biofluids and allow the assessment of disease-related changes in the proteome. This has made them a promising target for biomarker studies, especially in the field of neurodegeneration where access to diseased tissue is very limited. Genetic variants in the LRRK2 gene have been linked to both familial and sporadic forms of Parkinson's disease. With LRRK2 inhibitors entering clinical trials, there is an unmet need for biomarkers that reflect LRRK2-specific pathology and target engagement.

In this study, we used induced pluripotent stem cells derived from a patient with Parkinson's disease carrying the LRRK2 G2019S mutation and an isogenic gene-corrected control to generate human dopaminergic neurons. We isolated extracellular vesicles and neuronal cell lysates and characterized their proteomic signature using data-independent acquisition proteomics. Then, we performed differential expression analysis to identify dysregulated proteins in the mutated line. We used Metascape and gene ontology enrichment analysis on the dysregulated proteomes to identify changes in associated functional networks.

We identified 595 significantly differentially regulated proteins in extracellular vesicles and 3,205 in cell lysates. We visualized functionally relevant protein-protein interaction networks and identified key regulators within the dysregulated proteomes. Using gene ontology, we found a close association with biological processes relevant to neurodegeneration and Parkinson's disease. Finally, we focused on proteins that were dysregulated in both the extracellular and cellular proteomes. We provide a list of ten biomarker candidates that are functionally relevant to neurodegeneration and linked to LRRK2-associated pathology, for example, the sonic hedgehog signaling molecule, a protein that has tightly been linked to LRRK2-related disruption of cilia function.

In conclusion, we characterized the cellular and extracellular proteome of dopaminergic neurons carrying the LRRK2 G2019S mutation and proposed an experimentally based list of biomarker candidates for future studies.

Swelling, stiffness, and pain in synovial joints are primary hallmarks of osteoarthritis and rheumatoid arthritis. Hyperactivity of nociceptors and excessive release of inflammatory factors and pain mediators play a crucial role, with emerging data suggesting extensive remodelling and plasticity of joint innervations. Herein, we review structural, functional, and molecular alterations in sensory and autonomic axons wiring arthritic joints and revisit mechanisms implicated in the sensitization of nociceptors, leading to chronic pain. Sprouting and reorganization of sensory and autonomic fibers with the invasion of ectopic branches into surrounding inflamed tissues are associated with the upregulation of pain markers. These changes are frequently complemented by a phenotypic switch of sensory and autonomic profiles and activation of silent axons, inferring homeostatic adjustments and reprogramming of innervations. Identifying critical molecular players and neurobiological mechanisms underpinning the rewiring and sensitization of joints is likely to elucidate causatives of neuroinflammation and chronic pain, assisting in finding new therapeutic targets and opportunities for interventions.

Oligodendrocyte progenitor cells (OPCs) are highly dynamic, widely distributed glial cells of the central nervous system (CNS) that are responsible for generating myelinating oligodendrocytes during development. By also generating new oligodendrocytes in the adult CNS, OPCs allow formation of new myelin sheaths in response to environmental and behavioral changes and play a crucial role in regenerating myelin following demyelination (remyelination). However, the rates of OPC proliferation and differentiation decline dramatically with aging, which may impair homeostasis, remyelination, and adaptive myelination during learning. To determine how aging influences OPCs, we generated a novel transgenic mouse line that expresses membrane-anchored EGFP under the endogenous promoter/enhancer of Matrilin-4 (Matn4-mEGFP) and performed high-throughput single-cell RNA sequencing, providing enhanced resolution of transcriptional changes during key transitions from quiescence to proliferation and differentiation across the lifespan. Comparative analysis of OPCs isolated from mice aged 30 to 720 days, revealed that aging induces distinct inflammatory transcriptomic changes in OPCs in different states, including enhanced activation of HIF-1α and Wnt pathways. Inhibition of these pathways in acutely isolated OPCs from aged animals restored their ability to differentiate, suggesting that this enhanced signaling may contribute to the decreased regenerative potential of OPCs with aging. This Matn4-mEGFP mouse line and single-cell mRNA datasets of cortical OPCs across ages help to define the molecular changes guiding their behavior in various physiological and pathological contexts.

Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation- relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs.

Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-α /IFN-γ stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism.

This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphometric approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

Cell behavior emerges from the intracellular distribution of properties like protrusion, contractility and adhesion. Thus, characteristic emergent rules of collective migration can arise from cell-cell contacts locally tweaking architecture - orchestrating self-regulation during development, wound healing, and cancer progression. The new Drosophila testis-nascent-myotube-system allows dissection of contact-dependent migration in vivo at high resolution. Here, we describe a process driving gap-closure during migration: Contact-mesenchymalization via the axon guidance factor Plexin A. This is crucial for testis myotubes to migrate as a continuous sheet, allowing normal sculpting-morphogenesis. Cells must stay filopodial and dynamically ECM-tethered near cell-cell contacts to spread while collectively moving. Our data suggest Semaphorin 1B acts as a Plexin A antagonist, fine-tuning activation. Our data reveal a contact-dependent mechanism to maintain sheet-integrity during migration, driving organ-morphogenesis using a highly conserved pathway. This is relevant for understanding mesenchymal organ-sculpting and gap-closure in migratory contexts like angiogenesis.

Due to the negative impact of Haemonchus contortus in the tropics and subtropics, the detection of serum protein profiles that occur in infected sheep is of high relevance for targeted selective treatment strategies (TST). Herein, we integrated proteomics with phenotypic traits to elucidate physiological mechanisms associated to H. contortus infection in susceptible (Dorper - D) and resistant (Santa Inês - S) sheep breeds. Naïve female lambs were infected with H. contortus third-stage larvae on day zero (D0), and samples were collected weekly, for 28 days. Feces were used for individual fecal egg counts (FEC) blood for packed cell volume (PCV) and serum for specific antibody quantification through ELISA. Sera was collected on D0 (-) and D21 (+), and analyzed using a LC-MS/MS based proteomics approach. FEC, PCV, and anti-H. contortus antibody levels confirmed the absence of infection on D0. On D28 there was a significant difference between the two breeds for logFEC means (D = 3774 and S = 3141, p=0.033) and PCV means (D = 16.3 % and S = 24.3 %, p=0.038). From a total of 754 proteins identified, 68 differentially abundant proteins (DAPs) were noted. Phosphopyruvate hydratase (ENO3) was a DAP in all comparisons, while S+ vs D+ and S- vs D- shared the highest number of DAPs (8). Each of the four experimental groups clustered separately in a principal component analysis (PCA) of protein profile. Among the DAPs, proteins associated with the innate and adaptive immune system were detected when comparing S- vs D- and S+ vs D+. In D-, some proteins were linked to stress response to handling, sampling and heat. Focusing on the consequences of infection in each breed, in the D+ vs D- comparison, upregulated proteins were associated with inflammation control and immune response, where downregulated proteins pointed to a negative impact of infection on tissue anabolism, compromising muscle growth and fat deposition. In the S+ vs S- comparison, upregulated proteins were related to immune response, while the downregulated proteins were possibly linked to muscular development and growth, impaired by infection. Collectively, it can be concluded that ENO3 regulation emerges as a potential factor underlying the differential immune response observed between Santa Inês and Dorper sheep infected with H. contortus. In turn, detected acute phase proteins (APPs) reinforce their relation with infection, inflammation and stress conditions, whereas THEMIS-like may contribute to the immune system in Dorper. GSDMD, Guanylate-binding protein and ACAN warrant further investigation as possible biomarkers for TST strategy development.

Brown adipose tissue (BAT) represents an evolutionary innovation enabling placental mammals to regulate body temperature through adaptive thermogenesis. Brown adipocytes are surrounded by a dense network of blood vessels and sympathetic nerves that support their development and thermogenic function. Cold exposure stimulates BAT thermogenesis through the coordinated induction of brown adipogenesis, angiogenesis, and sympathetic innervation. However, how these distinct processes are coordinated remains unclear. Here, we identify Slit guidance ligand 3 (Slit3) as a new niche factor that mediates the crosstalk among adipocyte progenitors, endothelial cells, and sympathetic nerves. We show that adipocyte progenitors secrete Slit3 which regulates both angiogenesis and sympathetic innervation in BAT and is essential for BAT thermogenesis in vivo. Proteolytic cleavage of Slit3 generates secreted Slit3-N and Slit3-C fragments, which activate distinct receptors to stimulate angiogenesis and sympathetic innervation, respectively. Moreover, we introduce bone morphogenetic protein-1 (Bmp1) as the first Slit protease identified in vertebrates. In summary, this study underscores the essential role of Slit3-mediated neurovascular network expansion in enabling cold-induced BAT adaptation. The co-regulation of neurovascular expansion by Slit3 fragments provides a bifurcated yet harmonized approach to ensure a synchronized response of BAT to environmental challenges. This study presents the first evidence that adipocyte progenitors regulate tissue innervation, revealing a previously unrecognized dimension of cellular interaction within adipose tissue.

Large-scale analysis of single-cell gene expression has revealed transcriptomically defined cell subclasses present throughout the primate neocortex with gene expression profiles that differ depending upon neocortical region. Here, we test whether the interareal differences in gene expression translate to regional specializations in the physiology and morphology of infragranular glutamatergic neurons by performing Patch-seq experiments in brain slices from the temporal cortex (TCx) and motor cortex (MCx) of the macaque. We confirm that transcriptomically defined extratelencephalically projecting neurons of layer 5 (L5 ET neurons) include retrogradely labeled corticospinal neurons in the MCx and find multiple physiological properties and ion channel genes that distinguish L5 ET from non-ET neurons in both areas. Additionally, while infragranular ET and non-ET neurons retain distinct neuronal properties across multiple regions, there are regional morpho-electric and gene expression specializations in the L5 ET subclass, providing mechanistic insights into the specialized functional architecture of the primate neocortex.

The anterior visceral endoderm (AVE) differs from the surrounding visceral endoderm (VE) in its migratory behavior and ability to restrict primitive streak formation to the opposite side of the mouse embryo. To characterize the molecular bases for the unique properties of the AVE, we combined single-cell RNA sequencing of the VE prior to and during AVE migration with phosphoproteomics, high-resolution live-imaging, and short-term lineage labeling and intervention. This identified the transient nature of the AVE with attenuation of "anteriorizing" gene expression as cells migrate and the emergence of heterogeneities in transcriptional states relative to the AVE's position. Using cell communication analysis, we identified the requirement of semaphorin signaling for normal AVE migration. Lattice light-sheet microscopy showed that Sema6D mutants have abnormalities in basal projections and migration speed. These findings point to a tight coupling between transcriptional state and position of the AVE and identify molecular controllers of AVE migration.

In developing brains, axons exhibit remarkable precision in selecting synaptic partners among many non-partner cells. Evolutionarily conserved teneurins are transmembrane proteins that instruct synaptic partner matching. However, how intracellular signaling pathways execute teneurins' functions is unclear. Here, we use in situ proximity labeling to obtain the intracellular interactome of a teneurin (Ten-m) in the Drosophila brain. Genetic interaction studies using quantitative partner matching assays in both olfactory receptor neurons (ORNs) and projection neurons (PNs) reveal a common pathway: Ten-m binds to and negatively regulates a RhoGAP, thus activating the Rac1 small GTPases to promote synaptic partner matching. Developmental analyses with single-axon resolution identify the cellular mechanism of synaptic partner matching: Ten-m signaling promotes local F-actin levels and stabilizes ORN axon branches that contact partner PN dendrites. Combining spatial proteomics and high-resolution phenotypic analyses, this study advanced our understanding of both cellular and molecular mechanisms of synaptic partner matching.

In esophageal adenocarcinoma, the presence of lymph node metastases predicts patients' survival even after curative resection. Currently, there is no highly accurate marker for detecting the presence of lymph node metastasis. The SEMA3F/NRP2 axis was initially characterized in axon guidance and recent evidence has revealed its significant involvement in lymphangiogenesis, angiogenesis, and carcinogenesis. Hence, the objective of this study was to elucidate the roles of SEMA3F and its receptor NRP2 in esophageal adenocarcinoma. We conducted an immunohistochemical evaluation of SEMA3F and NRP2 protein expression in 776 patients with esophageal adenocarcinoma who underwent Ivor-Lewis esophagectomy at the University Hospital of Cologne. Total and positive cancer cell counts were digitally analyzed using QuPath and verified by experienced pathologists to ensure accuracy. Positive expression was determined as a cell percentage exceeding the 50th percentile threshold. In our cohort, patients exhibiting SEMA3F positive expression experience significantly lower pT- and pN-stages. In contrast, positive NRP2 expression is associated with the presence of lymph node metastases. Survival analyses showed that the expression status of NRP2 had no impact on patient survival. However, SEMA3F positivity was associated with a favorable patient survival outcome (median OS: 38.9 vs. 26.5 months). Furthermore, SEMA3F could be confirmed as an independent factor for better patient survival in patients with early tumor stage (pT1N0-3: HR = 0.505, p = 0.014, pT1-4N0: HR = 0.664, p = 0.024, pT1N0: HR = 0.483, p = 0.040). In summary, SEMA3F emerges as an independent predictor for a favorable prognosis in patients with early-stage esophageal adenocarcinoma. Additionally, NRP2 expression is linked to a higher risk of lymph node metastases occurrence. We hypothesize that low SEMA3F expression could identify patients with early-stage tumors who might benefit from more aggressive treatment options or intensified follow-up. Furthermore, SEMA3F and its associated pathways should be explored as potential tumor-suppressing agents.

Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Exosomes are small extracellular vesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived exosomes (KDEs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEs in a well-established high-fat diet (HFD) mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEs through size exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of HFD KDEs compared to KDEs obtained from control mice on a regular diet (RD), including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle (EV) reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEs are retrogradely trafficked into the DRG neuron cell body. Overall, our study presents a potential novel mode of communication between keratinocytes and DRG neurons in the skin, revealing a possible role for KDEs in contributing to the axonal degeneration that underlies neuropathic pain in PDN. Moreover, this study presents potential therapeutic targets in the skin for developing more effective, disease-modifying, and better-tolerated topical interventions for patients suffering from PDN, one of the most common and untreatable peripheral neuropathies.