p140Cap regulates memory and synaptic plasticity through Src-mediated and citron-N-mediated actin reorganization.

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.

A signaling loop of REST, TSC2 and ß-catenin governs proliferation and function of PC12 neural cells.

The RE-1-specific silencing transcription factor (REST or NRSF) is a transcription repressor that orchestrates differentiation and also operates in differentiated neurons and neurosecretory cells (neural cells). Its role in proliferation has been investigated so far only in rapidly growing tumors, with conflicting results: suppression in non-neural tumors, stimulation in medulloblastomas. Working with two clones of chromaffin-neuronal PC12 cells, which express different levels of REST, and using genetic complementation and knockdown approaches, we show that REST also promotes proliferation in differentiated neural cells. Mechanistically, this occurs by a signaling pathway involving REST, the GTPase-activating protein tuberin (TSC2) and the transcription co-factor ß-catenin. In PC12 cells, raised expression of REST correlates with reduced TSC2 levels, nuclear accumulation and co-transcriptional activation of ß-catenin, and increased expression of its target oncogenes Myc and Ccnd1, which might account for the proliferation advantage and the distinct morphology. Rest transcription is also increased, unveiling the existence of a self-sustaining, feed-forward REST-TSC2-ß-catenin signaling loop that is also operative in another neural cell model, NT2/D1 cells. Transfection of REST, knockdown of TSC2 or forced expression of active ß-catenin recapitulated the biochemical, functional and morphological properties of the high-expressing REST clone in wild-type PC12 cells. Upregulation of REST promoted proliferation and phenotypic changes, thus hindering neurosecretion. The new REST-TSC2-ß-catenin signaling paradigm might have an important role in various aspects of neural cell physiology and pathology, including the regulation of proliferation and neurosecretion.

Rapamycin-sensitive signals control TCR/CD28-driven Ifng, Il4 and Foxp3 transcription and promoter region methylation.

The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2+ cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation.

The tuberous sclerosis complex: balancing proliferation and survival.

Mutations in genes encoding either hamartin [TSC1 (tuberous sclerosis complex 1)] or tuberin (TSC2) result in a multisystem disorder characterized by the development of benign tumours and hamartomas in several organs. The TSC1 and TSC2 proteins form a complex that lies at the crossroad of many signalling pathways integrating the energy status of the cell with signals induced by nutrients and growth factors. The TSC1/2 complex is a critical negative regulator of mTORC1 [mTOR (mammalian target of rapamycin) complex 1], and by that controls anabolic processes to promote cell growth, proliferation and survival. In the present paper, we review recent evidence highlighting the notion that the TSC1/2 complex simultaneously controls mTOR-dependent and mTOR-independent signals critical for the balancing of cell proliferation and cell death.

CBP and SRF co-regulate dendritic growth and synaptic maturation.

The CREB-binding protein (CBP) exerts tight control of developmental processes. Here, we investigated the consequences of its selective ablation in newborn neurons. Mice in which CBP was eliminated during neuronal differentiation showed perinatal death and defective diaphragm innervation. Adult-born neurons also showed impaired growth and maturation after inducible and restricted CBP loss in dentate gyrus neuroprogenitors. Consistent with these in vivo findings, cultured neurons displayed impaired outgrowth, immature spines, and deficient activity-dependent synaptic remodeling after CBP ablation. These deficits coincided with broad transcriptional changes affecting genes involved in neuronal growth and plasticity. The affected gene set included many predicted targets of both CBP and the serum response factor (SRF), an activity-regulated transcription factor involved in structural plasticity. Notably, increasing SRF activity in a CBP-independent manner ameliorated the transcriptional, synaptic, and growth defects. These results underscore the relevance of CBP-SRF interactions during neuronal outgrowth and synaptic maturation, and demonstrate that CBP plays an essential role in supporting the gene program underlying the last steps of neuronal differentiation, both during development and in the adult brain.

Maternal Immune Activation Delays Excitatory-to-Inhibitory Gamma-Aminobutyric Acid Switch in Offspring.

BACKGROUND: The association between maternal infection and neurodevelopmental defects in progeny is well established, although the biological mechanisms and the pathogenic trajectories involved have not been defined. METHODS: Pregnant dams were injected intraperitoneally at gestational day 9 with polyinosinic:polycytidylic acid. Neuronal development was assessed by means of electrophysiological, optical, and biochemical analyses. RESULTS: Prenatal exposure to polyinosinic:polycytidylic acid causes an imbalanced expression of the Na+-K+-2Cl- cotransporter 1 and the K+-Cl- cotransporter 2 (KCC2). This results in delayed gamma-aminobutyric acid switch and higher susceptibility to seizures, which endures up to adulthood. Chromatin immunoprecipitation experiments reveal increased binding of the repressor factor RE1-silencing transcription (also known as neuron-restrictive silencer factor) to position 509 of the KCC2 promoter that leads to downregulation of KCC2 transcription in prenatally exposed offspring. Interleukin-1 receptor type I knockout mice, which display braked immune response and no brain cytokine elevation upon maternal immune activation, do not display KCC2/Na+-K+-2Cl- cotransporter 1 imbalance when implanted in a wild-type dam and prenatally exposed. Notably, pretreatment of pregnant dams with magnesium sulfate is sufficient to prevent the early inflammatory state and the delay in excitatory-to-inhibitory switch associated to maternal immune activation. CONCLUSIONS: We provide evidence that maternal immune activation hits a key neurodevelopmental process, the excitatory-to-inhibitory gamma-aminobutyric acid switch; defects in this switch have been unequivocally linked to diseases such as autism spectrum disorder or epilepsy. These data open the avenue for a safe pharmacological treatment that may prevent the neurodevelopmental defects caused by prenatal immune activation in a specific pregnancy time window.

Lack of IL-1R8 in neurons causes hyperactivation of IL-1 receptor pathway and induces MECP2-dependent synaptic defects.

Inflammation modifies risk and/or severity of a variety of brain diseases through still elusive molecular mechanisms. Here we show that hyperactivation of the interleukin 1 pathway, through either ablation of the interleukin 1 receptor 8 (IL-1R8, also known as SIGIRR or Tir8) or activation of IL-1R, leads to up-regulation of the mTOR pathway and increased levels of the epigenetic regulator MeCP2, bringing to disruption of dendritic spine morphology, synaptic plasticity and plasticity-related gene expression. Genetic correction of MeCP2 levels in IL-1R8 KO neurons rescues the synaptic defects. Pharmacological inhibition of IL-1R activation by Anakinra corrects transcriptional changes, restores MeCP2 levels and spine plasticity and ameliorates cognitive defects in IL-1R8 KO mice. By linking for the first time neuronal MeCP2, a key player in brain development, to immune activation and demonstrating that synaptic defects can be pharmacologically reversed, these data open the possibility for novel treatments of neurological diseases through the immune system modulation.

SNAP-25, a Known Presynaptic Protein with Emerging Postsynaptic Functions.

A hallmark of synaptic specializations is their dependence on highly organized complexes of proteins that interact with each other. The loss or modification of key synaptic proteins directly affects the properties of such networks, ultimately impacting synaptic function. SNAP-25 is a component of the SNARE complex, which is central to synaptic vesicle exocytosis, and, by directly interacting with different calcium channels subunits, it negatively modulates neuronal voltage-gated calcium channels, thus regulating intracellular calcium dynamics. The SNAP-25 gene has been associated with distinct brain diseases, including Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia and bipolar disorder, indicating that the protein may act as a shared biological substrate among different "synaptopathies". The mechanisms by which alterations in SNAP-25 may concur to these psychiatric diseases are still undefined, although alterations in neurotransmitter release have been indicated as potential causative processes. This review summarizes recent work showing that SNAP-25 not only controls exo/endocytic processes at the presynaptic terminal, but also regulates postsynaptic receptor trafficking, spine morphogenesis, and plasticity, thus opening the possibility that SNAP-25 defects may contribute to psychiatric diseases by impacting not only presynaptic but also postsynaptic functions.

Nanostructured TiO2 surfaces promote polarized activation of microglia, but not astrocytes, toward a proinflammatory profile.

Activation of glial cells, including astrocytes and microglia, has been implicated in the inflammatory responses underlying brain injury and neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The classic activation state (M1) is characterized by high capacity to present antigens, high production of nitric oxide (NO) and reactive oxygen species (ROS) and proinflammatory cytokines. Classically activated cells act as potent effectors that drive the inflammatory response and may mediate detrimental effects on neural cells. The second phenotype (M2) is an alternative, apparently beneficial, activation state, more related to a fine tuning of inflammation, scavenging of debris, promotion of angiogenesis, tissue remodeling and repair. Specific environmental chemical signals are able to induce these different polarization states. We provide here evidence that nanostructured substrates are able, exclusively in virtue of their physical properties, to push microglia toward the proinflammatory activation phenotype, with an efficacy which reflects the graded nanoscale rugosity. The acquisition of a proinflammatory phenotype appears specific for microglia and not astrocytes, indicating that these two cell types, although sharing common innate immune responses, respond differently to external physical stimuli.

SNAP-25 regulates spine formation through postsynaptic binding to p140Cap.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a member of the Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNARE) protein family, required for exocytosis of synaptic vesicles and regulation of diverse ion channels. Here, we show that acute reduction of SNAP-25 expression leads to an immature phenotype of dendritic spines that are, consistently, less functional. Conversely, over-expression of SNAP-25 results in an increase in the density of mature, Postsynaptic Density protein 95 (PSD-95)-positive spines. The regulation of spine morphogenesis by SNAP-25 depends on the protein's ability to bind both the plasma membrane and the adaptor protein p140Cap, a key protein regulating actin cytoskeleton and spine formation. We propose that SNAP-25 allows the organization of the molecular apparatus needed for spine formation by recruiting and stabilizing p140Cap.