p140Cap regulates the composition and localization of the NMDAR complex in synaptic lipid rafts.

The N-Methyl-D-aspartate receptors (NMDAR) are key players in both physiological and pathological synaptic plasticity because of their involvement in many aspects of neuronal transmission as well as learning and memory. The contribution in these events of different types of GluN2A-interacting proteins is still unclear. The p140Cap scaffold protein acts as a hub for postsynaptic complexes relevant to psychiatric and neurological disorders and regulates synaptic functions like the stabilization of mature dendritic spine, memory consolidation, long-term potentiation, and depression. Here we demonstrate that p140Cap directly binds the GluN2A subunit of NMDAR and modulates GluN2A-associated molecular network. Indeed, in p140Cap knockout male mice, GluN2A is less associated with PSD95 both in ex vivo synaptosomes and in cultured hippocampal neurons and p140Cap expression in knockout neurons can rescue GluN2A and PSD95 colocalization. p140Cap is crucial in the recruitment of GluN2A-containing NMDARs and, consequently, in regulating NMDARs intrinsic properties. p140Cap is associated to synaptic lipid-raft (LR) and to soluble postsynaptic membranes and GluN2A and PSD95 are less recruited into synaptic LR of p140Cap knockout male mice. g-STED microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in LR in an activity-dependent fashion. In the synaptic compartment p140Cap influences the association between GluN2A and PSD95 and modulates GluN2A enrichment into LR. Overall, such increase in these membrane domains rich in signalling molecules results in improved signal transduction efficiency.SIGNIFICANT STATEMENTHere we originally show that the adaptor protein p140Cap directly binds the GluN2A subunit of NMDAR and modulates the GluN2A-associated molecular network. Moreover, we show for the first time that p140Cap also associates to synaptic lipid rafts and controls the selective recruitment of GluN2A and PSD95 to this specific compartment. Finally, g-STED microscopy on hippocampal neurons confirmed that p140Cap is required for embedding GluN2A clusters in lipid rafts in an activity-dependent fashion. Overall, our findings provide the molecular and functional dissection of p140Cap as a new active member of a highly dynamic synaptic network involved in memory consolidation, LTP and LTD that are known to be altered in neurological and psychiatric disorders.

The p140Cap adaptor protein as a molecular hub to block cancer aggressiveness.

The p140Cap adaptor protein is a scaffold molecule encoded by the SRCIN1 gene, which is physiologically expressed in several epithelial tissues and in the neurons. However, p140Cap is also strongly expressed in a significant subset of cancers including breast cancer and neuroblastoma. Notably, cancer patients with high p140Cap expression in their primary tumors have a lower probability of developing a distant event and ERBB2-positive breast cancer sufferers show better survival. In neuroblastoma patients, SRCIN1 mRNA levels represent an independent risk factor, which is inversely correlated to disease aggressiveness. Consistent with clinical data, SRCIN1 gain or loss of function mouse models demonstrated that p140Cap may affect tumor growth and metastasis formation by controlling the signaling pathways involved in tumorigenesis and metastatic features. This study reviews data showing the relevance of SRCIN1/p140Cap in cancer patients, the impact of SRCIN1 status on p140Cap expression, the specific mechanisms through which p140Cap can limit cancer progression, the molecular functions regulated by p140Cap, along with the p140Cap interactome, to unveil its key role for patient stratification in clinics.

p140Cap Regulates GABAergic Synaptogenesis and Development of Hippocampal Inhibitory Circuits.

The neuronal scaffold protein p140Cap was investigated during hippocampal network formation. p140Cap is present in presynaptic GABAergic terminals and its genetic depletion results in a marked alteration of inhibitory synaptic activity. p140Cap-/- cultured neurons display higher frequency of miniature inhibitory postsynaptic currents (mIPSCs) with no changes of their mean amplitude. Consistent with a potential presynaptic alteration of basal GABA release, p140Cap-/- neurons exhibit a larger synaptic vesicle readily releasable pool, without any variation of single GABAA receptor unitary currents and number of postsynaptic channels. Furthermore, p140Cap-/- neurons show a premature and enhanced network synchronization and appear more susceptible to 4-aminopyridine-induced seizures in vitro and to kainate-induced seizures in vivo. The hippocampus of p140Cap-/- mice showed a significant increase in the number of both inhibitory synapses and of parvalbumin- and somatostatin-expressing interneurons. Specific deletion of p140Cap in forebrain interneurons resulted in increased susceptibility to in vitro epileptic events and increased inhibitory synaptogenesis, comparable to those observed in p140Cap-/- mice. Altogether, our data demonstrate that p140Cap finely tunes inhibitory synaptogenesis and GABAergic neurotransmission, thus regulating the establishment and maintenance of the proper hippocampal excitatory/inhibitory balance.

Human mesenchymal stem cells and derived extracellular vesicles induce regulatory dendritic cells in type 1 diabetic patients.

AIMS/HYPOTHESIS: Mesenchymal stem cells (MSCs) can exert an immunosuppressive effect on any component of the immune system, including dendritic cells (DCs), by direct contact, the release of soluble markers and extracellular vesicles (EVs). We evaluated whether MSCs and MSC-derived EVs have an immunomodulatory effect on monocyte-derived DCs in type 1 diabetes. METHODS: Bone marrow derived MSCs were characterised and EVs were obtained by ultracentrifugation. DCs were differentiated from CD14(+) cells, obtained from nine type 1 diabetic patients at disease onset, pulsed with antigen GAD65 and cultured with MSCs or EVs. Levels of DC maturation and activation markers were evaluated by flow cytometry. GAD65-pulsed DCs and autologous CD14(-) cell were co-cultured and IFN-γ enzyme-linked immunosorbent spot responses were assayed. Secreted cytokine levels were measured and Th17 and regulatory T cells were analysed. RESULTS: MSC- and EV-conditioned DCs acquired an immature phenotype with reduced levels of activation markers and increased IL-10 and IL-6 production. Conditioned DC plus T cell co-cultures showed significantly decreased IFN-γ spots and secretion levels. Moreover, higher levels of TGF-ß, IL-10 and IL-6 were detected compared with unconditioned DC plus T cell co-cultures. Conditioned DCs decreased Th17 cell numbers and IL-17 levels, and increased FOXP3(+) regulatory T cell numbers. EVs were internalised by DCs and EV-conditioned DCs exhibited a similar effect. CONCLUSIONS/INTERPRETATION: In type 1 diabetes, MSCs induce immature IL-10-secreting DCs in vitro, thus potentially intercepting the priming and amplification of autoreactive T cells in tissue inflammation. These DCs can contribute to the inhibition of inflammatory T cell responses to islet antigens and the promotion of the anti-inflammatory, regulatory responses exerted by MSCs.

p140Cap inhibits ß-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response.

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of ß-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of ß-Catenin depends on its ability to localize in and stabilize the ß-Catenin destruction complex, promoting enhanced ß-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the ß-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.

Exploring chitosan-shelled nanobubbles to improve HER2 + immunotherapy via dendritic cell targeting.

Immunotherapy is a valuable approach to cancer treatment as it is able to activate the immune system. However, the curative methods currently in clinical practice, including immune checkpoint inhibitors, present some limitations. Dendritic cell vaccination has been investigated as an immunotherapeutic strategy, and nanotechnology-based delivery systems have emerged as powerful tools for improving immunotherapy and vaccine development. A number of nanodelivery systems have therefore been proposed to promote cancer immunotherapy. This work aims to design a novel immunotherapy nanoplatform for the treatment of HER2 + breast cancer, and specially tailored chitosan-shelled nanobubbles (NBs) have been developed for the delivery of a DNA vaccine. The NBs have been functionalized with anti-CD1a antibodies to target dendritic cells (DCs). The NB formulations possess dimensions of approximately 300 nm and positive surface charge, and also show good physical stability up to 6 months under storage at 4 °C. In vitro characterization has confirmed that these NBs are capable of loading DNA with good encapsulation efficiency (82%). The antiCD1a-functionalized NBs are designed to target DCs, and demonstrated the ability to induce DC activation in both human and mouse cell models, and also elicited a specific immune response that was capable of slowing tumor growth in mice in vivo. These findings are the proof of concept that loading a tumor vaccine into DC-targeted chitosan nanobubbles may become an attractive nanotechnology approach for the future immunotherapeutic treatment of cancer.

p140Cap Controls Female Fertility in Mice Acting via Glutamatergic Afference on Hypothalamic Gonadotropin-Releasing Hormone Neurons.

p140Cap, encoded by the gene SRCIN1 (SRC kinase signaling inhibitor 1), is an adaptor/scaffold protein highly expressed in the mouse brain, participating in several pre- and post-synaptic mechanisms. p140Cap knock-out (KO) female mice show severe hypofertility, delayed puberty onset, altered estrus cycle, reduced ovulation, and defective production of luteinizing hormone and estradiol during proestrus. We investigated the role of p140Cap in the development and maturation of the hypothalamic gonadotropic system. During embryonic development, migration of Gonadotropin-Releasing Hormone (GnRH) neurons from the nasal placode to the forebrain in p140Cap KO mice appeared normal, and young p140Cap KO animals showed a normal number of GnRH-immunoreactive (-ir) neurons. In contrast, adult p140Cap KO mice showed a significant loss of GnRH-ir neurons and a decreased density of GnRH-ir projections in the median eminence, accompanied by reduced levels of GnRH and LH mRNAs in the hypothalamus and pituitary gland, respectively. We examined the number of kisspeptin (KP) neurons in the rostral periventricular region of the third ventricle, the number of KP-ir fibers in the arcuate nucleus, and the number of KP-ir punctae on GnRH neurons but we found no significant changes. Consistently, the responsiveness to exogenous KP in vivo was unchanged, excluding a cell-autonomous defect on the GnRH neurons at the level of KP receptor or its signal transduction. Since glutamatergic signaling in the hypothalamus is critical for both puberty onset and modulation of GnRH secretion, we examined the density of glutamatergic synapses in p140Cap KO mice and observed a significant reduction in the density of VGLUT-ir punctae both in the preoptic area and on GnRH neurons. Our data suggest that the glutamatergic circuitry in the hypothalamus is altered in the absence of p140Cap and is required for female fertility.

The N-terminal domain of the adaptor protein p140Cap interacts with Tiam1 and controls Tiam1/Rac1 axis.

The p140Cap adaptor protein, encoded by the SRCIN1 gene, negatively controls tumor progression, as demonstrated in the subgroup of HER2-amplified breast cancers and in neuroblastoma patients, where high p140Cap expression predicts a decreased probability of developing metastasis, with a significantly prolonged survival. In NeuT mice, a preclinical model or Her2-positive breast cancer, we previously reported that p140Cap counteracts Her2-dependent breast cancer progression, associating with the specific Rac1 Guanine Nucleotide Exchange Factor, Tiam1, and limiting the activation of both Tiam1 and Rac1. Here, we show that in TUBO breast cancer cells derived from the NeuT tumors, p140Cap expression causes Tiam1 redistribution along the apicobasal junctional axis. Furthermore, p140Cap and Tiam1 interact with E-cadherin, a member of the adherence junction, with a concomitant increase of E-cadherin at the cell membrane. We characterized biochemically the interaction between p140Cap and Tiam1, showing that the amino terminal region of p140Cap (1-287 amino acids) is sufficient to associate with full length Tiam1, and with the truncated catalytic domain of Tiam1, with a concomitant decrease of the Tiam1 activity. Moreover, in a large cohort of Her2 positive breast cancer, high levels of SRCIN1 expression positively correlates with increased survival in patients with high TIAM1 expression. Overall, our findings sustain a protective role of p140Cap in Her2 positive breast cancer, where p140Cap can associate with Tiam1 and negatively regulate the Tiam1/Rac1 axis.

The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma.

Neuroblastoma is the most common extra-cranial pediatric solid tumor, responsible for 13-15% of pediatric cancer death. Its intrinsic heterogeneity makes it difficult to target for successful therapy. The adaptor protein p140Cap/SRCIN1 negatively regulates tumor cell features and limits breast cancer progression. This study wish to assess if p140Cap is a key biological determinant of neuroblastoma outcome. RNAseq profiles of a large cohort of neuroblastoma patients show that SRCIN1 mRNA levels are an independent risk factor inversely correlated to disease aggressiveness. In high-risk patients, CGH+SNP microarray analysis of primary neuroblastoma identifies SRCIN1 as frequently altered by hemizygous deletion, copy-neutral loss of heterozygosity, or disruption. Functional experiments show that p140Cap negatively regulates Src and STAT3 signaling, affects anchorage-independent growth and migration, in vivo tumor growth and spontaneous lung metastasis formation. p140Cap also increases sensitivity of neuroblastoma cells to doxorubicin and etoposide treatment, as well as to a combined treatment with chemotherapy drugs and Src inhibitors. Our functional findings point to a causal role of p140Cap in curbing the aggressiveness of neuroblastoma, due to its ability to impinge on specific molecular pathways, and to sensitize cells to therapeutic treatment. This study provides the first evidence that the SRCIN1/p140Cap adaptor protein is a key player in neuroblastoma as a new independent prognostic marker for patient outcome and treatment. Altogether, these data highlight the potential clinical impact of SRCIN1/p140Cap expression in neuroblastoma tumors, in terms of reducing cytotoxic effects of chemotherapy, one of the main issues for pediatric tumor treatment.

Correction to: The SRCIN1/p140Cap adaptor protein negatively regulates the aggressiveness of neuroblastoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Dissecting the Shared and Context-Dependent Pathways Mediated by the p140Cap Adaptor Protein in Cancer and in Neurons.

The p140Cap adaptor protein is a scaffold molecule physiologically expressed in few epithelial tissues, such as the mammary gland, and in differentiated neurons. While the role of p140Cap in mammary gland epithelia is not still understood, we already know that a significant subset of breast cancers express p140Cap. In the subgroup of ERBB2-amplified breast cancers, a high p140Cap status predicts a significantly lower probability of developing a distant event and a clear difference in survival. p140Cap is causal in dampening ERBB2-positive tumor cell progression, impairing tumor onset and growth, and counteracting epithelial mesenchymal transition, resulting in decreased metastasis formation. Since only a few p140Cap interacting proteins have been identified in breast cancer and the molecular complexes and pathways underlying the cancer function of p140Cap are largely unknown, we generated a p140Cap interactome from ERBB2-positive breast cancer cells, identifying cancer specific components and those shared with the synaptic interactome. We identified 373 interacting proteins in cancer cells, including those with functions relevant to cell adhesion, protein homeostasis, regulation of cell cycle and apoptosis, which are frequently deregulated in cancer. Within the interactome, we identified 15 communities (clusters) with topology-functional relationships. In neurons, where p140Cap is key in regulating synaptogenesis, synaptic transmission and synaptic plasticity, it establishes an extensive interactome with proteins that cluster to sub complexes located in the postsynaptic density. p140Cap interactors converge on key synaptic processes, including synaptic transmission, actin cytoskeleton remodeling and cell-cell junction organization. Comparing the breast cancer to the synaptic interactome, we found 39 overlapping proteins, a relatively small overlap. However, cell adhesion and remodeling of actin cytoskeleton clearly emerge as common terms in the shared subset. Thus, the functional signature of the two interactomes is primarily determined by organ/tissue and functional specificity, while the overlap provides a list of shared functional terms, which might be linked to both cancer and neurological functions.

Direct and Intestinal Epithelial Cell-Mediated Effects of TLR8 Triggering on Human Dendritic Cells, CD14+CD16+ Monocytes and γδ T Lymphocytes.

Toll-like receptor (TLR)7/8 plays a crucial role in host recognition/response to viruses and its mucosal expression directly correlates with intestinal inflammation. The aim of this study was to investigate the role of TLR7/8 stimulation of intestinal epithelium in shaping the phenotype and functions of innate immunity cell subsets, and to define direct and/or epithelial cell-mediated mechanisms of the TLR7/8 agonist R848 immunomodulatory activity. We describe novel, TLR8-mediated, pro- and anti-inflammatory effects of R848 on ex vivo cultured human blood monocytes and γδ T lymphocytes, either induced by direct immune cell stimulation or mediated by intestinal epithelial cells (IEC). Apical stimulation with R848 led to its transport across normal polarized epithelial cell monolayer and resulted in the inhibition of monocyte differentiation toward immunostimulatory dendritic cells and Th1 type response. Furthermore, γδ T lymphocyte activation was promoted following direct exposure of these cells to the agonist. Conversely, a selective enrichment of the CD14+CD16+ monocyte subpopulation was observed, which required a CCL2-mediated inflammatory response of normal epithelial cells to R848. Of note, a TLR-mediated activation of control γδ T lymphocytes was promoted by inflamed intestinal epithelium from active Crohn's disease patients. This study unravels a novel regulatory mechanism linking the activation of the TLR8 pathway in IEC to the monocyte-mediated inflammatory response, and highlights the capacity of the TLR7/8 agonist R848 to directly enhance the activation of γδ T lymphocytes. Overall these results expand the range of cell targets and immune responses controlled by TLR8 triggering that may contribute to the antiviral response, to chronic inflammation, as well as to the adjuvant activity of TLR8 agonists, highlighting the role of intestinal epithelium microenvironment in shaping TLR agonist-induced responses.

Synaptic Interactome Mining Reveals p140Cap as a New Hub for PSD Proteins Involved in Psychiatric and Neurological Disorders.

Altered synaptic function has been associated with neurological and psychiatric conditions including intellectual disability, schizophrenia and autism spectrum disorder (ASD). Amongst the recently discovered synaptic proteins is p140Cap, an adaptor that localizes at dendritic spines and regulates their maturation and physiology. We recently showed that p140Cap knockout mice have cognitive deficits, impaired long-term potentiation (LTP) and long-term depression (LTD), and immature, filopodia-like dendritic spines. Only a few p140Cap interacting proteins have been identified in the brain and the molecular complexes and pathways underlying p140Cap synaptic function are largely unknown. Here, we isolated and characterized the p140Cap synaptic interactome by co-immunoprecipitation from crude mouse synaptosomes, followed by mass spectrometry-based proteomics. We identified 351 p140Cap interactors and found that they cluster to sub complexes mostly located in the postsynaptic density (PSD). p140Cap interactors converge on key synaptic processes, including transmission across chemical synapses, actin cytoskeleton remodeling and cell-cell junction organization. Gene co-expression data further support convergent functions: the p140Cap interactors are tightly co-expressed with each other and with p140Cap. Importantly, the p140Cap interactome and its co-expression network show strong enrichment in genes associated with schizophrenia, autism, bipolar disorder, intellectual disability and epilepsy, supporting synaptic dysfunction as a shared biological feature in brain diseases. Overall, our data provide novel insights into the molecular organization of the synapse and indicate that p140Cap acts as a hub for postsynaptic complexes relevant to psychiatric and neurological disorders.