Sorting nexin17 (SNX17) is a member of the sorting nexin family, which plays a crucial role in endosomal trafficking. Previous research has shown that SNX17 is involved in the recycling or degradation of various proteins associated with neurodevelopmental and neurological diseases in cell models. However, the significance of SNX17 in neurological function in the mouse brain has not been thoroughly investigated. In this study, we generated Snx17 knockout mice and observed that the homozygous deletion of Snx17 (Snx17-/-) resulted in lethality. On the other hand, heterozygous mutant mice (Snx17+/-) exhibited anxiety-like behavior with a reduced preference for social novelty. Furthermore, Snx17 haploinsufficiency led to impaired synaptic transmission and reduced maturation of dendritic spines. Through GST pulldown and interactome analysis, we identified the SRC kinase inhibitor, p140Cap, as a potential downstream target of SNX17. We also demonstrated that the interaction between p140Cap and SNX17 is crucial for dendritic spine maturation. Together, this study provides the first in vivo evidence highlighting the important role of SNX17 in maintaining neuronal function, as well as regulating social novelty and anxiety-like behaviors.

UNASSIGNED: Repeated cocaine administration changes histone acetylation and methylation on Lys residues and Deoxyribonucleic acid (DNA) within the nucleus accumbens (NAc). Recently Nestler\'s group explored histone Arg (R) methylation in reward processing models. Damez-Werno et al. (2016) reported that during human investigations and animal self-administration experiments, the histone mark protein-R-methyltransferase-6 (PRMT6) and asymmetric dimethylation of R2 on histone H3 (H3R2me2a) decreased in the rodent and cocaine-dependent human NAc. Overexpression of PRMT6 in D2-MSNs in all NAc neurons increased cocaine seeking, whereas PRMT6 overexpression in D1-MSNs protects against cocaine-seeking.
UNASSIGNED: The hypothesis is that dopaminylation (H3R2me2a binding) occurs in psychostimulant use disorder (PSU), and the binding inhibitor Srcin1, like the major DRD2 A2 allelic polymorphism, protects against psychostimulant seeking behavior by normalizing nucleus accumbens (NAc) dopamine expression.
UNASSIGNED: Numerous publications confirmed the association between the DRD2 Taq A1 allele (30-40 lower D2 receptor numbers) and severe cocaine dependence. Lepack et al. (2020) found that acute cocaine increases dopamine in NAc synapses, and results in histone H3 glutamine 5 dopaminylation (H3Q5dop) and consequent inhibition of D2 expression. The inhibition increases with chronic cocaine use and accompanies cocaine withdrawal. They also found that the Src kinase signaling inhibitor 1 (Srcin1 or p140CAP) during cocaine withdrawal reduced H3R2me2a binding. Consequently, this inhibited dopaminylation induced a \"homeostatic brake.\"
UNASSIGNED: The decrease in Src signaling in NAc D2-MSNs, (like the DRD2 Taq A2 allele, a well-known genetic mechanism protective against SUD) normalizes the NAc dopamine expression and decreases cocaine reward and motivation to self-administer cocaine. The Srcin1 may be an important therapeutic target.

The NMDARs are key players in both physiological and pathologic 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 neurologic disorders and regulates synaptic functions, such as the stabilization of mature dendritic spine, memory consolidation, LTP, and LTD. Here we demonstrate that p140Cap directly binds the GluN2A subunit of NMDAR and modulates GluN2A-associated molecular network. Indeed, in p140Cap KO male mice, GluN2A is less associated with PSD95 both in ex vivo synaptosomes and in cultured hippocampal neurons, and p140Cap expression in KO 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 KO male mice. Gated-stimulated emission depletion 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 signaling molecules results in improved signal transduction efficiency.SIGNIFICANCE STATEMENT Here 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, gated-stimulated emission depletion 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, which are known to be altered in neurologic and psychiatric disorders.

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 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 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.

OBJECTIVE: We previously demonstrated that progesterone (P4) interacted with folic acid (FA) and abolished the FA-reduced endothelial cell proliferation and migration. These findings led us to investigate whether FA can interfere with the P4-promoted breast cancer cell proliferation and migration.
METHODS: We conducted MTT and wound healing assay to evaluate cell proliferation and migration, respectively. Western blot analysis and immunoprecipitation were performed to examine the protein expression and protein-protein interaction, respectively.
RESULTS: We demonstrated that P4 promoted proliferation and migration of breast cancer cell lines (T47D, MCF-7, BT474, and BT483). However, co-treatment with P4 and FA together abolished these promotion effects. Treatment with P4 alone increased the formation of PR-cSrc complex and the phosphorylation of cSrc at tyrosine 416 (Tyr416). However, co-treatment with P4 and FA together increased the formations of cSrc-p140Cap, cSrc-Csk, and cSrc-p-Csk complex, and the phosphorylation of cSrc at tyrosine 527 (Tyr527). Co-treatment with P4 and FA together also abolished the activation of cSrc-mediated signaling pathways involved in the P4-promoted breast cancer cell proliferation and migration.
CONCLUSIONS: Co-treatment with FA and P4 together abolished the P4-promoted breast cancer cell proliferation and migration through decreasing the formation of PR-cSrc complex and increasing the formations of cSrc-p140Cap and cSrc-Csk complex, subsequently activating Csk, which in turn suppressed the phosphorylation of cSrc at Tyr416 and increased the phosphorylation of cSrc at Tyr527, hence inactivating the cSrc-mediated signaling pathways. The findings from this study might provide a new strategy for preventing the P4-promoted breast cancer progress.

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.

A major challenge in the neuroscience field is the identification of molecules and pathways that control synaptic plasticity and memory. Dendritic spines play a pivotal role in these processes, as the major sites of excitatory synapses in neuronal communication. Previous studies have shown that the scaffold protein p140Cap localizes into dendritic spines and that its knockdown negatively modulates spine shape in culture. However, so far, there is no information on its in vivo relevance. By using a knock-out mouse model, we here demonstrate that p140Cap is a key element for both learning and synaptic plasticity. Indeed, p140Cap(-/-) mice are impaired in object recognition test, as well as in LTP and in LTD measurements. The in vivo effects of p140Cap loss are presumably attenuated by noncell-autonomous events, since primary neurons obtained from p140Cap(-/-) mice show a strong reduction in number of mushroom spines and abnormal organization of synapse-associated F-actin. These phenotypes are most likely caused by a local reduction of the inhibitory control of RhoA and of cortactin toward the actin-depolymerizing factor cofilin. These events can be controlled by p140Cap through its capability to directly inhibit the activation of Src kinase and by its binding to the scaffold protein Citron-N. Altogether, our results provide new insight into how protein associated with dynamic microtubules may regulate spine actin organization through interaction with postsynaptic density components.

p140Cap is an adaptor protein that negatively controls tumor cell properties, by inhibiting in vivo tumor growth and metastasis formation. Our previous data demonstrated that p140Cap interferes with tumor growth and impairs invasive properties of cancer cells inactivating signaling pathways, such as the tyrosine kinase Src or E-cadherin/EGFR cross-talk. In breast cancer p140Cap expression inversely correlates with tumor malignancy. p140Cap is composed of several conserved domains that mediate association with specific partners. Here we focus our attention on two domains of p140Cap, the TER (Tyrosine Enriched Region) which includes several tyrosine residues, and the CT (Carboxy Terminal) which contains a proline rich sequence, involved in binding to SH2 and SH3 domains, respectively. By generating stable cell lines expressing these two proteins, we demonstrate that both TER and CT domains maintain the ability to associate the C-terminal Src kinase (Csk) and Src, to inhibit Src activation and Focal adhesion kinase (Fak) phosphorylation, and to impair in vitro and in vivo tumor cell features. In particular expression of TER and CT proteins in cancer cells inhibits in vitro and in vivo growth and directional migration at a similar extent of the full length p140Cap protein. Moreover, by selective point mutations and deletion we show that the ability of the modules to act as negative regulators of cell migration and proliferation mainly resides on the two tyrosines (Y) inserted in the EPLYA and EGLYA sequences in the TER module and in the second proline-rich stretch contained in the CT protein. Gene signature of cells expressing p140Cap, TER or CT lead to the identification of a common pattern of 105 down-regulated and 128 up-regulated genes, suggesting that the three proteins can act through shared pathways. Overall, this work highlights that the TER and CT regions of p140Cap can efficiently suppress tumor cell properties, opening the perspective that short, defined p140Cap regions can have therapeutic effects.