Neuronal Guidance Research Articles

The neurorepellent SLIT2 inhibits LPS-induced proinflammatory signaling in macrophages

Abstract Macrophages are important mediators of immune responses with critical roles in the recognition and clearance of pathogens, as well as in the resolution of inflammation and wound healing. The neuronal guidance cue SLIT2 has been widely studied for its effects on immune cell functions, most notably directional cell migration. Recently, SLIT2 has been shown to directly enhance bacterial killing by macrophages, but the effects of SLIT2 on inflammatory activation of macrophages are less known. Using RNA sequencing analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay, we determined that in murine bone marrow–derived macrophages challenged with the potent proinflammatory mediator lipopolysaccharide (LPS), exposure to the bioactive N-terminal fragment of SLIT2 (NSLIT2) suppressed production of proinflammatory cytokines interleukin (IL)-6 and IL-12 and concurrently increased the anti-inflammatory cytokine IL-10. We found that NSLIT2 inhibited LPS-induced MyD88- and TRIF-mediated signaling cascades and did not inhibit LPS-induced internalization of Toll-like receptor 4 (TLR4), but instead inhibited LPS-induced upregulation of macropinocytosis. Inhibition of macropinocytosis in macrophages attenuated LPS-induced production of proinflammatory IL-6 and IL-12 and concurrently enhanced anti-inflammatory IL-10. Taken together, our results indicate that SLIT2 can selectively modulate macrophage response to potent proinflammatory stimuli, such as LPS, by attenuating proinflammatory activation and simultaneously enhancing anti-inflammatory activity. Our results highlight the role of macropinocytosis in proinflammatory activation of macrophages exposed to LPS. Given that LPS-producing bacteria cause host illness through synergistic direct bacterial infection and excessive LPS-induced systemic inflammation, our work suggests a novel therapeutic role for SLIT2 in combatting the significant morbidity and mortality of patients with Gram-negative bacterial sepsis.

Mossy Fiber Sprouting in Temporal Lobe Epilepsy: The Impact of Netrin-1, DCC, and Gene Expression Changes.

Temporal lobe epilepsy (TLE) is the most common form of drug-resistant epilepsy, often associated with hippocampal sclerosis (HS), which involves selective neuronal loss in the Cornu Ammonis subregion 1 CA1 and CA4 regions of the hippocampus. Granule cells show migration and mossy fiber sprouting, though the mechanisms remain unclear. Microglia play a role in neurogenesis and synaptic modulation, suggesting they may contribute to epilepsy. This study examines the role of microglia and axonal guidance molecules in neuronal reorganization in TLE. Nineteen hippocampal samples from patients with TLE undergoing epilepsy surgery were analyzed. Microglial activity (M1/M2-like microglia) and neuronal guidance molecules were assessed using microscopy and semi-automated techniques. Gene expression was evaluated using the nCounter Expression Profiling method. Neuronal cell loss was correlated with decreased activity of the M1 microglial phenotype. In the CA2 region, neuronal preservation was linked to increased mossy fiber sprouting and microglial presence. Neuronal markers such as Deleted in Colorectal Cancer (DCC) and Synaptopodin were reduced in areas of cell death, while Netrin-1 was elevated in the granule cell layer, potentially influencing mossy fiber sprouting. The nCounter analysis revealed downregulation of genes involved in neuronal activity (e.g., NPAS4, BCL-2, GRIA1) and upregulation of IκB, indicating reduced neuroinflammation. This study suggests reduced neuroinflammation in areas of neuronal loss, while regions with preserved neurons showed mossy fiber sprouting associated with microglia, Netrin-1, and DCC.

Understanding Aβ25-35 peptide altered exosomal proteome and associated pathways linked with the Alzheimer's disease pathogenesis using human neuroblastoma SH-SY5Y Cells.

The central nervous system (CNS) involves a complex interplay of communications between the neurons and various glial cells, which is crucial for brain functions. The major interactomes are exosomes that transmit sundry molecules (DNA, miRNAs, and proteins) between the cells and thus alter cell physiology. Exosomes can act as neuroprotective or neurodegenerative agents depending on the microenvironment of cells secreting them. Therefore, revealing exosome proteome becomes important to understand donor cells' physiology and its effect on the recipient cell. In this study, oxidative stress was induced by Aβ25-35 in the human neuroblastoma SH-SY5Y cells and the protective effects of phytochemical ferulic acid (FA) were evaluated alone and in combination with Aβ25-35 (pre-treated for 3h before Aβ25-35 exposure) and proteome of their secreted exosomes was analyzed, which was carried out via a high-resolution LC-MS Triple-ToF and further network-based analysis has been carried out using various bioinformatics tools. The proteomic profiling enlightened the multiple roles of exosomes as proteins associated with the various pathways advocate that exosomes can mediate a wide range of effects, from normal physiological processes like synaptic plasticity, neuronal metabolic support, nerve regeneration, DNA repair, axon guidance, and long-term potentiation (LTP) to abnormal pathological processes like inflammatory responses, oxidative stress, apoptosis, and formation of neutrophil extracellular traps (NETs). On comparison, treatment with Aβ25-35 resulted in a significant modulation of the exosomal proteome, promoting pathways associated with neurodegeneration. Conversely, the phytochemical FA displayed a protective effect by effectively countering Aβ25-35-induced oxidative stress responses linked with neurodegeneration, as seen in Alzheimer's disease (AD). Taken together, this study highlights the dual role of exosomes in physiological and pathophysiological neurodegenerative AD, which intricately depend on the particular cellular milieu.

Glu592 of the axon guidance receptor ROBO3 mediates a pH-dependent interaction with NELL2 ligand.

There are only a few studies on the function of neuronal axon guidance molecules during low brain pH conditions. We previously reported that roundabout (ROBO) 2, a receptor for the axon guidance molecule SLIT, can bind to the neural epidermal growth factor-like-like (NELL) ligands in acidic conditions by conformational change of its ectodomain. Here, we show that the ROBO3 receptor also exhibits a pH-dependent increase in binding to the NELL2 ligand. We found that the Glu592 residue of ROBO3 at the binding interface between NELL2 and ROBO3 is a pH sensor and that the formation of a new hydrogen bonding network, due to protonation of the Glu592, leads to increased binding in acidic conditions. These results suggest that NELL2-ROBO3 signaling could be regulated by extracellular pH.

Elevated Netrin-4 Expression and Its Action in Infrapatellar Fat Pad.

Knee osteoarthritis (KOA) is a degenerative joint disease characterized by inflammation and cartilage degradation. The infrapatellar fat pad (IFP), located beneath the patella within the knee joint, serves as a key anatomical structure involved in cushioning and supporting the knee. It is also an active endocrine organ that secretes various bioactive substances, potentially influencing the local inflammatory environment and contributing to KOA pathogenesis. Netrin-4 (NTN4), a protein primarily known for its role in neuronal guidance, has been implicated in various non-neuronal functions, including inflammatory processes and tissue remodeling. This study aims to explore the involvement of NTN4 in KOA, focusing on its expression in the IFP and its potential impact on disease progression. This study involved 82 patients with radiographically confirmed KOA undergoing total knee arthroplasty (TKA). The correlation between NTN4 expression and OA pathology, including Kellgren-Lawrence (K/L) grades, was investigated. NTN4-expressing cells were identified in the stromal vascular fraction, including fibroblastic, hematopoietic, and endothelial cells of the IFP. To elucidate the molecular effects of NTN4, RNA sequencing (RNA-seq) was performed on fibroblastic cells treated with recombinant NTN4. Subsequent quantitative PCR (qPCR) was used to validate the RNA-seq findings. NTN4 expression was significantly elevated in the IFP of patients with advanced KOA (K/L grades 3 and 4) compared to those with early-stage disease (K/L grade 2). Higher NTN4 expression was found in fibroblastic cells, and RNA-seq analysis revealed upregulation of genes associated with pro-inflammatory pathways, including IL-17 and TNF-α signaling, and matrix degradation. Notably, genes including IL6, MMP1, CXCL1, and CXCL8 were significantly elevated, as confirmed by qPCR, indicating NTN4's role in promoting an inflammatory and catabolic environment. Our findings suggest that NTN4 plays a significant role in the pathogenesis of KOA by promoting inflammation and matrix degradation within the IFP. Although NTN4 expression was not directly correlated with clinical symptoms, its elevated expression in fibroblastic cells and influence on inflammatory and degradative pathways suggest a potential mechanism for exacerbating joint damage. Targeting NTN4 could offer a novel therapeutic approach to mitigating inflammation and slowing disease progression in KOA, ultimately improving patient outcomes. Further research is needed to clarify NTN4's specific roles and therapeutic potential in OA management.

Plexin C1 influences immune response to intracellular LPS and survival in murine sepsis

BackgroundIntracellular sensing of lipopolysaccharide (LPS) is essential for the immune response against gram-negative bacteria and results in activation of caspase-11 and pyroptotic cell death with fatal consequences in sepsis. We found the neuronal guidance receptor plexin C1 (PLXNC1) influences the intracellular response to LPS.MethodsWe employed a murine model of sepsis via cecal ligation and binding (CLP), using PLXNC1-/- mice and littermate controls, and additionally transfected murine bone-marrow-derived macrophages (BMDMs) from both genotypes with LPS to achieve activation of the noncanonical inflammasome ex vivo. Additionally, we transfected the PLXNC1 ligand SL4c-d in vivo and ex vivo to examine its effect on intracellular LPS response.ResultsWe found the neuronal guidance receptor PLXNC1 dampens the intracellular response to LPS by interacting with adenylate cyclase 4 (ADCY4) and protein kinase A activity, which in turn diminishes caspase-11 expression. The absence of PLXNC1 results in excessive inflammation marked by increased cytokine release, increased secondary organ injury and reduced sepsis survival in a murine sepsis model induced by CLP. Notably, administration of SL4c-d—peptide ligand of PLXNC1—reduces the inflammatory response during CLP-induced sepsis and improves survival.ConclusionsThese results elucidate a previously unknown mechanism for PLXNC1 suppressing excessive noncanonical inflammasome activity and offer a new potential target for treatment of sepsis with its detrimental effects.

MicroRNAs Associated with IgLON Cell Adhesion Molecule Expression.

The IgLON family of cell adhesion molecules consists of five members (LSAMP, OPCML, neurotrimin, NEGR1, and IgLON5) discovered as supporters of neuronal development, axon growth and guidance, and synapse formation and maintenance. Tumour suppression properties have recently been emerging based on antiproliferative effects through the modulation of oncogenic pathways. Available evidence endorses a role for non-coding RNAs or microRNAs as relevant controllers of IgLON molecule expression that can impact their critical physiological and pathological roles. Current findings support a function for long non-coding RNAs and microRNAs in the modulation of LSAMP expression in cell senescence, cancer biogenesis, addiction, and pulmonary hypertension. For OPCML, data point to a role for several microRNAs in the control of tumorigenesis. MicroRNAs were detected in neurotrimin-mediated functions in cancer biogenesis and in Schwann cell responses to peripheral nerve injury. For NEGR1, studies have mainly investigated microRNA involvement in neuronal responses to ischaemic injury, although data also exist about tumorigenesis and endothelial cell dysfunction. For IgLON5, information is only available about microRNA involved in myocardial infarction. In conclusion, despite much information being still missing and further research needed, the emerging picture favours a model in which non-coding RNAs exert a crucial role in modulating IgLON expression, ultimately affecting their important physiological functions.

Nuclear receptor subfamily 4A2: Novel role and a potential therapeutic lead in cancers.

e18017 Background: Perineural invasion (PNI) is a pathological process where cancer cells infiltrate surrounding nerve tissues, markedly increasing the risk of cancer recurrence, metastasis, post-surgical complications, and associated pain. Alarmingly, PNI is prevalent in 80 - 100% of Head and Neck Squamous Carcinoma (HNSCC) and Pancreatic Ductal Adenocarcinoma (PDAC) cases. In these tumors, nerve invasion often occurs as a distinct route of metastasis, bypassing the usual lymphatic or vascular routes. Despite the rigor of surgical interventions and post-operative radiation, PNI-associated tumors recurrently manifest, emphasizing an urgent need to elucidate the underlying molecular interplay between tumor cells and nerves. Methods: Our study focuses on the Nuclear receptor subfamily 4A2 (NR4A2) transcription factor, known for its pivotal roles in cellular homeostasis and response to pathological conditions. Results: Comprehensive analyses employing tissue microarrays, curated public datasets, and cell lines unveiled a consistent overexpression of NR4A2 in HNSCC and PDAC tumor cells compared to their normal counterparts. Intriguingly, genome-wide chromatin immunoprecipitation (ChIP) sequencing with an NR4A2-specific antibody highlighted an enrichment of genes linked to neuronal guidance, suggesting a probable NR4A2-mediated mechanism underpinning PNI. Further, an in-depth analysis of these ChIP-enriched neuronal genes using The Cancer Genome Atlas (TCGA) confirmed their heightened expression in HNSCC and PDAC tumor tissues relative to healthy tissues. Advancing our research with graph neural networks and sophisticated bioinformatics, we identified two potent lead compounds targeting NR4A2. Preliminary Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) assessments of these compounds indicate favorable safety and efficacy profiles for clinical application. We are currently conducting comprehensive evaluations of these compounds, deciphering their molecular mode of action and therapeutic potential in cell culture and animal models. Conclusions: To sum up, our findings reveal a yet unknown role of NR4A2 in the landscape of PNI in HNSCC and PDAC. The AI-driven discovery and ensuing validation of NR4A2-centric inhibitors present a novel therapeutic avenue to curb cancer progression, prevent metastasis, and potentially improve patient outcomes.

Cerebrospinal fluid proteomic profile of frailty: Results from the PROLIPHYC cohort.

Frailty is a clinical state reflecting a decrease in physiological reserve capacities, known to affect numerous biological pathways and is associated with health issues, including neurodegenerative diseases. However, how global protein expression is affected in the central nervous system in frail subject remains underexplored. In this post hoc cross-sectional biomarker analysis, we included 90 adults (52-85 years) suspected of normal pressure hydrocephalus (NPH) and presenting with markers of neurodegenerative diseases. We investigated the human proteomic profile of cerebrospinal fluid associated with frailty defined by an established cumulated frailty index (FI, average = 0.32), not enriched for neurology clinical features. Using a label-free quantitative proteomic approach, we identified and quantified 999 proteins of which 13 were positively associated with frailty. Pathway analysis with the top positively frailty-associated proteins revealed enrichment for proteins related to inflammation and immune response. Among the 60 proteins negatively associated with frailty, functional pathways enriched included neurogenesis, synaptogenesis and neuronal guidance. We constructed a frailty prediction model using ridge regression with 932 standardized proteins. Our results showed that the "proteomic model" could become an equivalent predictor of FI in order to study chronological age. This study represents the first comprehensive exploration of the proteomic profile of frailty within cerebrospinal fluid. It sheds light on the physiopathology of frailty, particularly highlighting processes of neuroinflammation and inhibition of neurogenesis. Our findings unveil a range of biological mechanisms that are dysregulated in frailty, in NPH subjects at risk of neurodegenerative impairment, offering new perspectives on frailty phenotyping and prediction.

A mismatch in the expression of cell surface molecules induces tissue-intrinsic defense against aberrant cells

Tissue-intrinsic error correction enables epithelial cells to detect abnormal neighboring cells and facilitate their removal from the tissue. One of these pathways, "interface surveillance," is triggered by cells with aberrant developmental and cell-fate-patterning pathways. It remains unknown which molecular mechanisms provide cells with the ability to compare fate between neighboring cells. We demonstrate that Drosophila imaginal discs express an array of cell surface molecules previously implicated in neuronal axon guidance processes. They include members of the Robo, Teneurin, Ephrin, Toll-like, or atypical cadherin families. Importantly, a mismatch in expression levels of these cell surface molecules between adjacent cells is sufficient to induce interface surveillance, indicating that differences in expression levels between neighboring cells, rather than their absolute expression levels, are crucial. Specifically, a mismatch in Robo2 and Robo3, but not Robo1, induces enrichment of actin, myosin II, and Ena/Vasp, as well as activation of JNK and apoptosis at clonal interfaces. Moreover, Robo2 can induce interface surveillance independently of its cytosolic domain and without the need for the Robo-ligand Slit. The expression of Robo2 and other cell surface molecules, such as Teneurins or the Ephrin receptor is regulated by fate-patterning pathways intrinsic and extrinsic to the wing disc, as well as by expression of oncogenic RasV12. Combined, we demonstrate that neighboring cells respond to a mismatch in surface code patterns mediated by specific transmembrane proteins and reveal a novel function for these cell surface proteins in cell fate recognition and removal of aberrant cells during development and homeostasis of epithelial tissues.

Cancer-mediated axonal guidance of sensory neurons in a microelectrode-based innervation MPS

Despite recent advances in the field of microphysiological systems (MPSs), availability of models capable of mimicking the interactions between the nervous system and innervated tissues is still limited. This represents a significant challenge in identifying the underlying processes of various pathological conditions, including neuropathic, cardiovascular and metabolic disorders. In this novel study, we introduce a compartmentalized three-dimensional (3D) coculture system that enables physiologically relevant tissue innervation while recording neuronal excitability. By integrating custom microelectrode arrays into tailored glass chips microfabricated via selective laser-etching, we developed an entirely novel class of innervation MPSs (INV-MPS). This INV-MPS allows for manipulation, visualization, and electrophysiological analysis of individual axons innervating complex 3D tissues. Here, we focused on sensory innervation of 3D tumor tissue as a model case study since cancer-induced pain represents a major unmet medical need. The system was compared with existing nociception models and successfully replicated axonal chemoattraction mediated by nerve growth factor (NGF). Remarkably, in the absence of NGF, 3D cancer spheroids cocultured in the adjacent compartment induced sensory neurons to consistently cross the separating barrier and establish fine innervation. Moreover, we observed that crossing sensory fibers could be chemically excited by distal application of known pain-inducing agonists only when cocultured with cancer cells. To our knowledge, this is the first system showcasing morphological and electrophysiological analysis of 3D-innervated tumor tissue in vitro, paving the way for a plethora of studies into innervation-related diseases and improving our understanding of underlying pathophysiology.

Abstract B074: Novel targeting of semaphorin 7a-mediated endocrine therapy resistance in an immune competent ER+ breast cancer model

Abstract Estrogen receptor-positive (ER+) breast cancer (BC) comprises over 70% of all breast cancers and is the leading cause of BC-related deaths in women worldwide. Despite available therapies against ER+ BC, recurrence inevitably arises primarily due to therapeutic resistance. Our objective is to improve treatment strategies for patients with ER+ BC by studying the therapeutic targeting potential of semaphorin-7a (SEMA7A)-mediated endocrine therapy resistance in an immune competent model of ER+ breast cancer. It is known that SEMA7A, a neuronal guidance protein that is dysegulated in several cancer types, confers poor prognosis and survival in BC patients, is regulated by ER, activates integrin-mediated PI3K/AKT signaling and promotes resistance to the ER-targeted therapies. In the normal mammary gland, SEMA7A binds β1- and a6-integrin to activate pathways including pro-survival PI3K/AKT signaling, which is known to promote tumor cell survival and multi-drug resistance. Using reverse phase protein array we identified differential expression between empty vector (EV) or SEMA7A-overexpressing (OE) MCF7 cells. SEMA7A OE cells had increased survival signaling proteins (pAKT, pS6K, pPTEN, pFAK, pSrc, pmTOR). Other protein types upregulated included cell adhesion (E-Cadherin), signal transduction (ITGB1, STAT3, ITGB4, NFkB, ILK1) and regulation of cell death/survival (BCL2, RB1, Annexin1). We validated that SEMA7A-induced signaling activates AKT in ER+ BC cells, and that inhibition of PI3K via alpelisib (Novartis) and GCT-007 (Global Cancer Technology) reduces tumor growth in vitro and in vivo in a MCF7/NCG xenograft model. Our preliminary studies show that SEMA7A OE tumors in NCG mice respondly poorly to fulvestrant, while EV tumors are sensitive. Further, PI3K inhibition by alpelisib or GCT-007 rendered SEMA7A OE, fulvestrant-resistant tumors sensitive to treatment in this model (n=5/group). Our lab has also created an anti-SEMA7A monoclonal antibody (SmAb) that directly targets SEMA7A. Our results show that SmAb inhibits tumor growth in triple negative mouse BC mouse models, but is less effective in immunocompromised models. Since our data show that SmAb restores anti-tumor immunity, we sought to inhibit the SEMA7A-integrin-PI3K/AKT pathway in an immune competent ER+ model. Therefore, utilized a novel syngeneic ER+ preclinical model comprised of TC11 murine tumor cells, which express SEMA7A, and FVB/N mice. Mice were treated with tamoxifen, and after tumors did not respond, PI3K inhibitors were initiated, which slowed tumor growth but did not achieve full regression. Our findings show that the addition of either PI3K inhibitor (alpelisib or GCT-007) to tamoxifen was more effective than tamoxifen alone. Future studies will investigate the combinations with SmAb and identify immune-related effects after PI3K/SEMA7A inhibition. If effective, our studies would suggest that ER+ SEMA7A+ patient tumors should be considered for PI3K/SEMA7A-targeted therapies. Citation Format: Rachel N Steinmetz, Lyndsey S Crump, Traci R Lyons. Novel targeting of semaphorin 7a-mediated endocrine therapy resistance in an immune competent ER+ breast cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B074.

Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling

The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis invitro and invivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.

Transcriptomic analyses of rats exposed to chronic mild stress: Modulation by chronic treatment with the antipsychotic drug lurasidone

Exposure to stressful experiences accounts for almost half of the risk for mental disorders. Hence, stress-induced alterations represent a key target for pharmacological interventions aimed at restoring brain function in affected individuals. We have previously demonstrated that lurasidone, a multi-receptor antipsychotic drug approved for the treatment of schizophrenia and bipolar depression, can normalize the functional and molecular impairments induced by stress exposure, representing a valuable tool for the treatment of stress-induced mental illnesses. However, the mechanisms that may contribute to the therapeutic effects of lurasidone are still poorly understood. Here, we performed a transcriptomic analysis on the prefrontal cortex (PFC) of adult male rats exposed to the chronic mild stress (CMS) paradigm and we investigated the impact of chronic lurasidone treatment on such changes. We found that CMS exposure leads to an anhedonic phenotype associated with a down-regulation of different pathways associated to neuronal guidance and synaptic plasticity within the PFC. Interestingly, a significant part of these alterations (around 25%) were counteracted by lurasidone treatment. In summary, we provided new insights on the transcriptional changes relevant for the therapeutic intervention with lurasidone, which may ultimately promote resilience.

THU580 A Subset Of Migrating Cells Co-express Gonadotropin Releasing Hormone And Prokineticin Receptor 2 During Embryonic Development

Abstract Disclosure: A.E. Danku: None. C.F. Elias: None. A subset of migrating cells co-express gonadotropin releasing hormone and prokineticin receptor 2 during embryonic development Antoinette Danku and Carol F Elias. Neuroscience Graduate Program and Department of Molecular & Integrative Physiology, University to Michigan. Reproductive vitality in humans is strongly influenced by developmental foundations. The successful migration of GnRH cells and the pulsatile secretion of gonadotropin releasing hormone (GnRH) drives pubertal onset and dictates long-term fertility outcomes. During development, GnRH neurons migrate from the nasal placode to the basal forebrain to reach their destination in the medial preoptic area (MPOA). Aberrant GnRH migration causes redirection of axonal projections to atypical brain regions and dysregulated GnRH secretion. Disruption of GnRH neuronal function causes infertility or reproductive dysfunction. These etiologies are grouped as isolated gonadotropin deficiencies (IGD). Kallmann’s syndrome (KS) is a pediatric IGD that manifests as anosmia and hormonal dysfunction that can result in pubertal delay or infertility. Children with KS often have mutations in prokineticin 2 (Prok2) or the cognate receptor (ProkR2).Prok2 and ProkR2 are required for the formation of the olfactory bulb in rodents and humans. Loss-of-function mutations in these genes also disrupts GnRH cell migration, but whether the activation of ProkR2 regulates the guidance of GnRH neurons to their final destination has not been defined. Previous studies suggested that GnRH and ProkR2 are expressed in distinct cells, but how the prokineticin system may alter GnRH migration is largely unknown. To gain insights into the underlying mechanisms associated with ProkR2-GnRH interaction and migration, we conducted single nuclei RNA sequencing in nasal-forebrain junction of embryos at embryonic day 16 (E16) and fluorescent in situ hybridization in three developmental time points: E14, E16, and E18 for validation and spatial evaluation. Using these methods, we were able to isolate GnRH and ProkR2 cells and sort their cell types into clusters. While these cells differ in their expression profiles, they both express genes associated with migration at E16. Our findings suggest that ProkR2 cells in the nasal-forebrain junction migrate along with GnRH cells. Additionally, a subset of these migrating cells co-express ProkR2 and GnRH mRNA. Further studies are needed to determine whether this subset of cells take on specialized roles in GnRH cell migration and maturation. Presentation: Thursday, June 15, 2023

Netrin-1 and RGMa: Novel Regulators of Atherosclerosis-Related Diseases.

Neuronal guidance proteins (NGPs) havebeen demonstrated toguide the elongation of neuronal axonal growth cones in the developing central nervous system. Non-neuronal functions of NGPs have also been described, especially in relation to atherosclerosis. Netrin-1 and repulsive guidance molecule a (RGMa) are NGPs that have been shown to regulate endothelial cell adhesion and angiogenesis, macrophage migration and apoptosis, smooth muscle cells (SMCs) phenotypic dedifferentiation and mobility, chemokine activities, and inflammatory responses during atherosclerosis initiation and progression. However, mechanistic studies have generated controversy about the specific role of Netrin-1 in atherosclerosis due to the diversity of its structure, receptors and cell sources, and the actions of RGMa in atherosclerosis have not been reported in previous reviews. Therefore, the current work reviews the evidence for roles of Netrin-1 and RGMa in the initiation and progression of atherosclerosis and discusses potential therapeutic targets in the future.

Mechanical properties of the brain: Focus on the essential role of Piezo1-mediated mechanotransduction in the CNS.

The brain is a highly mechanosensitive organ, and changes in the mechanical properties of brain tissue influence many physiological and pathological processes. Piezo type mechanosensitive ion channel component 1 (Piezo1), a protein found in metazoans, is highly expressed in the brain and involved in sensing changes of the mechanical microenvironment. Numerous studies have shown that Piezo1-mediated mechanotransduction is closely related to glial cell activation and neuronal function. However, the precise role of Piezo1 in the brain requires further elucidation. This review first discusses the roles of Piezo1-mediated mechanotransduction in regulating the functions of a variety of brain cells, and then briefly assesses the impact of Piezo1-mediated mechanotransduction on the progression of brain dysfunctional disorders. Mechanical signaling contributes significantly to brain function. Piezo1-mediated mechanotransduction regulates processes such as neuronal differentiation, cell migration, axon guidance, neural regeneration, and oligodendrocyte axon myelination. Additionally, Piezo1-mediated mechanotransduction plays significant roles in normal aging and brain injury, as well as the development of various brain diseases, including demyelinating diseases, Alzheimer's disease, and brain tumors. Investigating the pathophysiological mechanisms through which Piezo1-mediated mechanotransduction affects brain function will give us a novel entry point for the diagnosis and treatment of numerous brain diseases.

Novel loss of function mutation in TUBA1A gene compromises tubulin stability and proteostasis causing spastic paraplegia and ataxia.

Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and β-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene.

A streptavidin–biotin system combined with magnetic actuators for remote neuronal guidance

The ability to control neuronal mobility and organization is of great importance in developing neuronal interfaces and novel therapeutic approaches. An emerging promising method is the manipulation of neuronal cells from afar via magnetic forces. Nevertheless, using magnetic iron oxide nanoparticles as internal actuators may lead to biotoxicity, adverse influence on intracellular processes, and thus requires prerequisite considerations for therapeutic approaches. Magnetizing the cells via the incorporation of magnetic particles that can be applied extracellularly is advantageous. Herein, we have developed a magnetic system based on streptavidin–biotin interaction to decorate cellular membrane with magnetic elements. In this model, superparamagnetic microparticles, coated with streptavidin, were specifically bound to biotinylated PC12 cells. We demonstrated that cell movement can be directed remotely by the forces produced by pre-designed magnetic fields. First, using time lapse imaging, we analyzed the kinetics of cell migration towards the higher flux zone. Next, to form organized networks of cells we designed and fabricated micro-patterned magnetic devices. The fabricated devices were composed of a variety of ferromagnetic shapes, sputter-deposited onto glass substrates. Cells that were conjugated to the magnetic particles were plated atop the micro-patterned substrates, attracted to the magnetic actuators and became fixed onto the magnetic patterns. In all, our study presents a novel system based on a well-known molecular technology combined with nanotechnology that may well lead to the expansion of implantable magnetic actuators to organize and direct cellular growth.