NaCl enhances CD8+ T cell effector functions in cancer immunotherapy.

CD8+ T cells control tumors but inevitably become dysfunctional in the tumor microenvironment. Here, we show that sodium chloride (NaCl) counteracts T cell dysfunction to promote cancer regression. NaCl supplementation during CD8+ T cell culture induced effector differentiation, IFN-γ production and cytotoxicity while maintaining the gene networks responsible for stem-like plasticity. Accordingly, adoptive transfer of tumor-specific T cells resulted in superior anti-tumor immunity in a humanized mouse model. In mice, a high-salt diet reduced the growth of experimental tumors in a CD8+ T cell-dependent manner by inhibiting terminal differentiation and enhancing the effector potency of CD8+ T cells. Mechanistically, NaCl enhanced glutamine consumption, which was critical for transcriptional, epigenetic and functional reprogramming. In humans, CD8+ T cells undergoing antigen recognition in tumors and predicting favorable responses to checkpoint blockade immunotherapy resembled those induced by NaCl. Thus, NaCl metabolism is a regulator of CD8+ T cell effector function, with potential implications for cancer immunotherapy.

Feeling depressed? Keep calm, and watch microglia.

The immune system actively regulates brain activity through the engagement of immune cells and immunomodulatory molecules. In this issue of Immunity, Klawonn et al. show that the activation of microglia in the striatum triggers an IL-6-mediated autocrine loop and the release of prostaglandins, which in turn induce a negative affective state via the stimulation of medium spiny neurons.

Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring.

Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Immune synaptopathies: how maternal immune activation impacts synaptic function during development.

In the last two decades, the term synaptopathy has been largely used to underline the concept that impairments of synaptic structure and function are the major determinant of brain disorders, including neurodevelopmental disorders. This notion emerged from the progress made in understanding the genetic architecture of neurodevelopmental disorders, which highlighted the convergence of genetic risk factors onto molecular pathways specifically localized at the synapse. However, the multifactorial origin of these disorders also indicated the key contribution of environmental factors. It is well recognized that inflammation is a risk factor for neurodevelopmental disorders, and several immune molecules critically contribute to synaptic dysfunction. In the present review, we highlight this concept, which we define by the term "immune-synaptopathy," and we discuss recent evidence suggesting a bi-directional link between the genetic architecture of individuals and maternal activation of the immune system in modulating brain developmental trajectories in health and disease.

Maternal immune activation leads to defective brain-blood vessels and intracerebral hemorrhages in male offspring.

Intracerebral hemorrhages are recognized risk factors for neurodevelopmental disorders and represent early biomarkers for cognitive dysfunction and mental disability, but the pathways leading to their occurrence are not well defined. We report that a single intrauterine exposure of the immunostimulant Poly I:C to pregnant mice at gestational day 9, which models a prenatal viral infection and the consequent maternal immune activation, induces the defective formation of brain vessels and causes intracerebral hemorrhagic events, specifically in male offspring. We demonstrate that maternal immune activation promotes the production of the TGF-ß1 active form and the consequent enhancement of pSMAD1-5 in males' brain endothelial cells. TGF-ß1, in combination with IL-1ß, reduces the endothelial expression of CD146 and claudin-5, alters the endothelium-pericyte interplay resulting in low pericyte coverage, and increases hemorrhagic events in the adult offspring. By showing that exposure to Poly I:C at the beginning of fetal cerebral angiogenesis results in sex-specific alterations of brain vessels, we provide a mechanistic framework for the association between intragravidic infections and anomalies of the neural vasculature, which may contribute to neuropsychiatric disorders.

Embryo-restricted responses to maternal IL-17A promote neurodevelopmental disorders in mouse offspring.

Prenatal imprinting to interleukin 17A (IL-17A) triggers behavioral disorders in offspring. However, reported models of maternal immune activation utilizing immunostimulants, lack specificity to elucidate the anatomical compartments of IL-17A's action and the distinct behavioral disturbances it causes. By combining transgenic IL-17A overexpression with maternal deficiency in its receptor, we established a novel model of prenatal imprinting to maternal IL-17A (acronym: PRIMA-17 model). This model allowed us to study prenatal imprinting established exclusively through embryo-restricted IL-17A responses. We demonstrated a transfer of transgenic IL-17A across the placental barrier, which triggered the development of selected behavioral deficits in mouse offspring. More specifically, embryonic responses to IL-17A resulted in communicative impairment in early-life measured by reduced numbers of nest retrieval calls. In adulthood, IL-17A-imprinted offspring displayed an increase in anxiety-like behavior. We advocate our PRIMA-17 model as a useful tool to study neurological deficits in mice.

Loss of interleukin 1 signaling causes impairment of microglia- mediated synapse elimination and autistic-like behaviour in mice.

In the last years, the hypothesis that elevated levels of proinflammatory cytokines contribute to the pathogenesis of neurodevelopmental diseases has gained popularity. IL-1 is one of the main cytokines found to be elevated in Autism spectrum disorder (ASD), a complex neurodevelopmental condition characterized by defects in social communication and cognitive impairments. In this study, we demonstrate that mice lacking IL-1 signaling display autistic-like defects associated with an excessive number of synapses. We also show that microglia lacking IL-1 signaling at early neurodevelopmental stages are unable to properly perform the process of synapse engulfment and display excessive activation of mammalian target of rapamycin (mTOR) signaling. Notably, even the acute inhibition of IL-1R1 by IL-1Ra is sufficient to enhance mTOR signaling and reduce synaptosome phagocytosis in WT microglia. Finally, we demonstrate that rapamycin treatment rescues the defects in IL-1R deficient mice. These data unveil an exclusive role of microglial IL-1 in synapse refinement via mTOR signaling and indicate a novel mechanism possibly involved in neurodevelopmental disorders associated with defects in the IL-1 pathway.

Pentraxin 3 regulates synaptic function by inducing AMPA receptor clustering via ECM remodeling and ß1-integrin.

Control of synapse number and function in the developing central nervous system is critical to the formation of neural circuits. Astrocytes play a key role in this process by releasing factors that promote the formation of excitatory synapses. Astrocyte-secreted thrombospondins (TSPs) induce the formation of structural synapses, which however remain post-synaptically silent, suggesting that completion of early synaptogenesis may require a two-step mechanism. Here, we show that the humoral innate immune molecule Pentraxin 3 (PTX3) is expressed in the developing rodent brain. PTX3 plays a key role in promoting functionally-active CNS synapses, by increasing the surface levels and synaptic clustering of AMPA glutamate receptors. This process involves tumor necrosis factor-induced protein 6 (TSG6), remodeling of the perineuronal network, and a ß1-integrin/ERK pathway. Furthermore, PTX3 activity is regulated by TSP1, which directly interacts with the N-terminal region of PTX3. These data unveil a fundamental role of PTX3 in promoting the first wave of synaptogenesis, and show that interplay of TSP1 and PTX3 sets the proper balance between synaptic growth and synapse function in the developing brain.

Correction: Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity.

[This corrects the article DOI: 10.1371/journal.ppat.1008654.].

Mutant prion proteins increase calcium permeability of AMPA receptors, exacerbating excitotoxicity.

Prion protein (PrP) mutations are linked to genetic prion diseases, a class of phenotypically heterogeneous neurodegenerative disorders with invariably fatal outcome. How mutant PrP triggers neurodegeneration is not known. Synaptic dysfunction precedes neuronal loss but it is not clear whether, and through which mechanisms, disruption of synaptic activity ultimately leads to neuronal death. Here we show that mutant PrP impairs the secretory trafficking of AMPA receptors (AMPARs). Specifically, intracellular retention of the GluA2 subunit results in synaptic exposure of GluA2-lacking, calcium-permeable AMPARs, leading to increased calcium permeability and enhanced sensitivity to excitotoxic cell death. Mutant PrPs linked to different genetic prion diseases affect AMPAR trafficking and function in different ways. Our findings identify AMPARs as pathogenic targets in genetic prion diseases, and support the involvement of excitotoxicity in neurodegeneration. They also suggest a mechanistic explanation for how different mutant PrPs may cause distinct disease phenotypes.

A novel SYN1 missense mutation in non-syndromic X-linked intellectual disability affects synaptic vesicle life cycle, clustering and mobility.

Intellectual Disability is a common and heterogeneous disorder characterized by limitations in intellectual functioning and adaptive behaviour, whose molecular mechanisms remain largely unknown. Among the numerous genes found to be involved in the pathogenesis of intellectual disability, 10% are located on the X-chromosome. We identified a missense mutation (c.236 C > G; p.S79W) in the SYN1 gene coding for synapsin I in the MRX50 family, affected by non-syndromic X-linked intellectual disability. Synapsin I is a neuronal phosphoprotein involved in the regulation of neurotransmitter release and neuronal development. Several mutations in SYN1 have been identified in patients affected by epilepsy and/or autism. The S79W mutation segregates with the disease in the MRX50 family and all affected members display intellectual disability as sole clinical manifestation. At the protein level, the S79W Synapsin I mutation is located in the region of the B-domain involved in recognition of highly curved membranes. Expression of human S79W Synapsin I in Syn1 knockout hippocampal neurons causes aberrant accumulation of small clear vesicles in the soma, increased clustering of synaptic vesicles at presynaptic terminals and increased frequency of excitatory spontaneous release events. In addition, the presence of S79W Synapsin I strongly reduces the mobility of synaptic vesicles, with possible implications for the regulation of neurotransmitter release and synaptic plasticity. These results implicate SYN1 in the pathogenesis of non-syndromic intellectual disability, showing that alterations of synaptic vesicle trafficking are one possible cause of this disease, and suggest that distinct mutations in SYN1 may lead to distinct brain pathologies.

Lack of Methyl-CpG Binding Protein 2 (MeCP2) Affects Cell Fate Refinement During Embryonic Cortical Development.

During differentiation, neurons progressively restrict their fate repressing the expression of specific genes. Here we describe the involvement in such developmental steps of the methyl-CpG binding protein 2 (MeCP2), an epigenetic factor that participates to chromatin folding and transcriptional regulation. We previously reported that, due to transcriptional impairments, the maturation of Mecp2 null neurons is delayed. To evaluate whether this could stem from altered progenitors proliferation and differentiation, we investigated whether lack of Mecp2 affects these features both in vitro and in vivo. We show that in Mecp2 null embryonic cortexes the expression of genes defining the identity of proliferating neuroprogenitors is enriched and that their permanence in the G1 phase is prolonged. Moreover, the number of cells transitioning from a stage of maturation to a more mature one is increased in Mecp2 null embryonic cortices, in line with the central role of G1 for cell identity refinement. We thus suggest that, possibly due to the lack of proper transcriptional control normally exerted by Mecp2, fate refinement is impaired in developing null cells. We propose that the maturation delay affecting the developing Mecp2 null cortex originates, at least in part, from deranged mechanisms of cell fate refinement.

Synapsin I deletion reduces neuronal damage and ameliorates clinical progression of experimental autoimmune encephalomyelitis.

The classical view of multiple sclerosis (MS) pathogenesis states that inflammation-mediated demyelination is responsible for neuronal damage and loss. However, recent findings show that impairment of neuronal functions and demyelination can be independent events, suggesting the coexistence of other pathogenic mechanisms. Due to the inflammatory milieu, subtle alterations in synaptic function occur, which are probably at the basis of the early cognitive decline that often precedes the neurodegenerative phases in MS patients. In particular, it has been reported that inflammation enhances excitatory synaptic transmission while it decreases GABAergic transmission in vitro and ex vivo. This evidence points to the idea that an excitation/inhibition imbalance occurs in the inflamed MS brain, even though the exact molecular mechanisms leading to this synaptic dysfunction are as yet not completely clear. Along this line, we observed that acute treatment of primary hippocampal neurons in culture with pro-inflammatory cytokines leads to an increased phosphorylation of synapsin I (SynI) by ERK1/2 kinase and to an increase in the frequency of spontaneous synaptic vesicle release events, which is prevented by SynI deletion. In vivo, the ablation of SynI expression is protective in terms of disease progression and neuronal damage in the experimental autoimmune encephalomyelitis mouse model of MS. Our results point to a possible key role in MS pathogenesis of the neuronal protein SynI, a regulator of excitation/inhibition balance in neuronal networks.

REST/NRSF-mediated intrinsic homeostasis protects neuronal networks from hyperexcitability.

Intrinsic homeostasis enables neuronal circuits to maintain activity levels within an appropriate range by modulating neuronal voltage-gated conductances, but the signalling pathways involved in this process are largely unknown. We characterized the process of intrinsic homeostasis induced by sustained electrical activity in cultured hippocampal neurons based on the activation of the Repressor Element-1 Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF). We showed that 4-aminopyridine-induced hyperactivity enhances the expression of REST/NRSF, which in turn, reduces the expression of voltage-gated Na(+) channels, thereby decreasing the neuronal Na(+) current density. This mechanism plays an important role in the downregulation of the firing activity at the single-cell level, re-establishing a physiological spiking activity in the entire neuronal network. Conversely, interfering with REST/NRSF expression impaired this homeostatic response. Our results identify REST/NRSF as a critical factor linking neuronal activity to the activation of intrinsic homeostasis and restoring a physiological level of activity in the entire neuronal network.

Defects During Mecp2 Null Embryonic Cortex Development Precede the Onset of Overt Neurological Symptoms.

MeCP2 is associated with several neurological disorders; of which, Rett syndrome undoubtedly represents the most frequent. Its molecular roles, however, are still unclear, and data from animal models often describe adult, symptomatic stages, while MeCP2 functions during embryonic development remain elusive. We describe the pattern and timing of Mecp2 expression in the embryonic neocortex highlighting its low but consistent expression in virtually all cells and show the unexpected occurrence of transcriptional defects in the Mecp2 null samples at a stage largely preceding the onset of overt symptoms. Through the deregulated expression of ionic channels and glutamatergic receptors, the lack of Mecp2 during early neuronal maturation leads to the reduction in the neuronal responsiveness to stimuli. We suggest that such features concur to morphological alterations that begin affecting Mecp2 null neurons around the perinatal age and become evident later in adulthood. We indicate MeCP2 as a key modulator of the transcriptional mechanisms regulating cerebral cortex development. Neurological phenotypes of MECP2 patients could thus be the cumulative result of different adverse events that are already present at stages when no obvious signs of the pathology are evident and are worsened by later impairments affecting the central nervous system during maturation and maintenance of its functionality.

Milestones to the Discovery of T-type Calcium Channel Blockers for the Treatment of Generalized Epilepsies.

We describe the discovery and optimization of new, brain-penetrant T-type calcium channel blockers. We present optimized compounds with excellent efficacy in a rodent model of generalized absence-like epilepsy. Along the fine optimization of a chemical series with a pharmacological target located in the CNS (target potency, brain penetration, and solubility), we successfully identified an Ames negative aminopyrazole as putative metabolite of this compound series. Our efforts culminated in the selection of compound 20, which was elected as a preclinical candidate.

Catechols as Sources of Hydrogen Atoms in Radical Deiodination and Related Reactions.

When used with trialkylboranes, catechol derivatives, which are low-cost and low toxicity, are valuable hydrogen atom donors for radical chain reactions involving alkyl iodides and related radical precursors. The system 4-tert-butylcatechol/triethylborane has been used to reduce a series of secondary and tertiary iodides, a xanthate, and a thiohydroxamate ester. Catechol derivatives are right in the optimal kinetic window for synthetic applications, as demonstrated by highly efficient radical cyclizations. Cyclizations leading to the formation of quaternary centers can be performed in an all-at-once process (no slow addition of the hydrogen atom donor) at standard concentrations. The H-donor properties of catechol derivatives can be fine-tuned by changing their substitution pattern. In slow radical cyclization processes, an enhanced ratio of cyclized/uncyclized products was obtained by using 3-methoxycatechol instead of 4-tert-butylcatechol.

Reduced SNAP-25 alters short-term plasticity at developing glutamatergic synapses.

SNAP-25 is a key component of the synaptic-vesicle fusion machinery, involved in several psychiatric diseases including schizophrenia and ADHD. SNAP-25 protein expression is lower in different brain areas of schizophrenic patients and in ADHD mouse models. How the reduced expression of SNAP-25 alters the properties of synaptic transmission, leading to a pathological phenotype, is unknown. We show that, unexpectedly, halved SNAP-25 levels at 13-14 DIV not only fail to impair synaptic transmission but instead enhance evoked glutamatergic neurotransmission. This effect is possibly dependent on presynaptic voltage-gated calcium channel activity and is not accompanied by changes in spontaneous quantal events or in the pool of readily releasable synaptic vesicles. Notably, synapses of 13-14 DIV neurons with reduced SNAP-25 expression show paired-pulse depression as opposed to paired-pulse facilitation occurring in their wild-type counterparts. This phenotype disappears with synapse maturation. As alterations in short-term plasticity represent a new mechanism contributing to cognitive impairments in intellectual disabilities, our data provide mechanistic clues for neuronal circuit alterations in psychiatric diseases characterized by reduced expression of SNAP-25.