Microglial TNFα orchestrates protein phosphorylation in the cortex during the sleep period and controls homeostatic sleep.

Sleep intensity is adjusted by the length of previous awake time, and under tight homeostatic control by protein phosphorylation. Here, we establish microglia as a new cellular component of the sleep homeostasis circuit. Using quantitative phosphoproteomics of the mouse frontal cortex, we demonstrate that microglia-specific deletion of TNFα perturbs thousands of phosphorylation sites during the sleep period. Substrates of microglial TNFα comprise sleep-related kinases such as MAPKs and MARKs, and numerous synaptic proteins, including a subset whose phosphorylation status encodes sleep need and determines sleep duration. As a result, microglial TNFα loss attenuates the build-up of sleep need, as measured by electroencephalogram slow-wave activity and prevents immediate compensation for loss of sleep. Our data suggest that microglia control sleep homeostasis by releasing TNFα which acts on neuronal circuitry through dynamic control of phosphorylation.

Heterotypic Amyloid ß interactions facilitate amyloid assembly and modify amyloid structure.

It is still unclear why pathological amyloid deposition initiates in specific brain regions or why some cells or tissues are more susceptible than others. Amyloid deposition is determined by the self-assembly of short protein segments called aggregation-prone regions (APRs) that favour cross-ß structure. Here, we investigated whether Aß amyloid assembly can be modified by heterotypic interactions between Aß APRs and short homologous segments in otherwise unrelated human proteins. Mining existing proteomics data of Aß plaques from AD patients revealed an enrichment in proteins that harbour such homologous sequences to the Aß APRs, suggesting heterotypic amyloid interactions may occur in patients. We identified homologous APRs from such proteins and show that they can modify Aß assembly kinetics, fibril morphology and deposition pattern in vitro. Moreover, we found three of these proteins upon transient expression in an Aß reporter cell line promote Aß amyloid aggregation. Strikingly, we did not find a bias towards heterotypic interactions in plaques from AD mouse models where Aß self-aggregation is observed. Based on these data, we propose that heterotypic APR interactions may play a hitherto unrealized role in amyloid-deposition diseases.

Nuclear import of the DSCAM-cytoplasmic domain drives signaling capable of inhibiting synapse formation.

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.

SorCS1-mediated sorting in dendrites maintains neurexin axonal surface polarization required for synaptic function.

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.

Trafficking mechanisms of synaptogenic cell adhesion molecules.

Nearly every aspect of neuronal function, from wiring to information processing, critically depends on the highly polarized architecture of neurons. Establishing and maintaining the distinct molecular composition of axonal and dendritic compartments requires precise control over the trafficking of the proteins that make up these cellular domains. Synaptic cell adhesion molecules (CAMs), membrane proteins with a critical role in the formation, differentiation and plasticity of synapses, require targeting to the correct pre- or postsynaptic compartment for proper functioning of neural circuits. However, the mechanisms that control the polarized trafficking, synaptic targeting, and synaptic abundance of CAMs are poorly understood. Here, we summarize current knowledge about the sequential trafficking events along the secretory pathway that control the polarized surface distribution of synaptic CAMs, and discuss how their synaptic targeting and abundance is additionally influenced by post-secretory determinants. The identification of trafficking-impairing mutations in CAMs associated with various neurodevelopmental disorders underscores the importance of correct protein trafficking for normal brain function.

Ghrelin triggers the synaptic incorporation of AMPA receptors in the hippocampus.

Ghrelin is a peptide mainly produced by the stomach and released into circulation, affecting energy balance and growth hormone release. These effects are guided largely by the expression of the ghrelin receptor growth hormone secretagogue type 1a (GHS-R1a) in the hypothalamus and pituitary. However, GHS-R1a is expressed in other brain regions, including the hippocampus, where its activation enhances memory retention. Herein we explore the molecular mechanism underlying the action of ghrelin on hippocampal-dependent memory. Our data show that GHS-R1a is localized in the vicinity of hippocampal excitatory synapses, and that its activation increases delivery of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic-type receptors (AMPARs) to synapses, producing functional modifications at excitatory synapses. Moreover, GHS-R1a activation enhances two different paradigms of long-term potentiation in the hippocampus, activates the phosphatidylinositol 3-kinase, and increases GluA1 AMPAR subunit and stargazin phosphorylation. We propose that GHS-R1a activation in the hippocampus enhances excitatory synaptic transmission and synaptic plasticity by regulating AMPAR trafficking. Our study provides insights into mechanisms that may mediate the cognition-enhancing effect of ghrelin, and suggests a possible link between the regulation of energy metabolism and learning.

Pediatric adrenal insufficiency: thirty years experience at a Portuguese hospital.

BACKGROUND: Adrenal insufficiency (AI) is a life-threatening condition caused by an impaired secretion of the adrenal glucocorticoid and mineralocorticoid hormones. It comprises a heterogeneous group of primary, secondary and acquired disorders. Presentation differs according to the child's age, but it usually presents with nonspecific and insidious symptoms and signs. The main purpose of this study was to describe and compare patients with primary or secondary AI. METHODS: Retrospective analysis of all patients with adrenal insufficiency followed at the Pediatric Endocrinology Unit in a tertiary care Portuguese hospital over the last 30 years. Data on family history, age at the first manifestation and at etiological diagnosis, and clinical presentation (symptoms, signs and laboratory evaluation) was gathered for all patients. RESULTS: Twenty-eight patients with AI were included; 67.9% were male, with a median (25th-75th percentile, P25-P75) age of 1 (0.5-36) month at the first presentation. The principal diagnostic categories were panhypopituitarism (42.9%) and congenital adrenal hyperplasia (25%). The most frequent manifestations (75%) were vomiting and weight loss. They were followed for a median (P25-P75) period of 3.5 (0.6-15.5) years. In respect to neurodevelopmental delay and learning difficulties, they were more common in the secondary AI group. CONCLUSIONS: Despite medical advances, the diagnosis and management of AI remains a challenge, particularly in the pediatric population, and clinicians must have a high index of suspicion. An early identification of AI can prevent a potential lethal outcome, which may result from severe cardiovascular and hemodynamic instability.

Ligand-independent activity of the ghrelin receptor modulates AMPA receptor trafficking and supports memory formation.

The biological signals of hunger, satiety, and memory are interconnected. The role of the hormone ghrelin in regulating feeding and memory makes ghrelin receptors attractive targets for associated disorders. We investigated the effects of the high ligand-independent activity of the ghrelin receptor GHS-R1a on the physiology of excitatory synapses in the hippocampus. Blocking this activity produced a decrease in the synaptic content of AMPA receptors in hippocampal neurons and a reduction in GluA1 phosphorylation at Ser845 Reducing the ligand-independent activity of GHS-R1a increased the surface diffusion of AMPA receptors and impaired AMPA receptor-dependent synaptic delivery induced by chemical long-term potentiation. Accordingly, we found that blocking this GHS-R1a activity impaired spatial and recognition memory in mice. These observations support a role for the ligand-independent activity of GHS-R1a in regulating AMPA receptor trafficking under basal conditions and in the context of synaptic plasticity that underlies learning.

A Modular Organization of LRR Protein-Mediated Synaptic Adhesion Defines Synapse Identity.

Pyramidal neurons express rich repertoires of leucine-rich repeat (LRR)-containing adhesion molecules with similar synaptogenic activity in culture. The in vivo relevance of this molecular diversity is unclear. We show that hippocampal CA1 pyramidal neurons express multiple synaptogenic LRR proteins that differentially distribute to the major excitatory inputs on their apical dendrites. At Schaffer collateral (SC) inputs, FLRT2, LRRTM1, and Slitrk1 are postsynaptically localized and differentially regulate synaptic structure and function. FLRT2 controls spine density, whereas LRRTM1 and Slitrk1 exert opposing effects on synaptic vesicle distribution at the active zone. All LRR proteins differentially affect synaptic transmission, and their combinatorial loss results in a cumulative phenotype. At temporoammonic (TA) inputs, LRRTM1 is absent; FLRT2 similarly controls functional synapse number, whereas Slitrk1 function diverges to regulate postsynaptic AMPA receptor density. Thus, LRR proteins differentially control synaptic architecture and function and act in input-specific combinations and a context-dependent manner to specify synaptic properties.

Neuronal Polarity: MAP2 Shifts Secretory Vesicles into High Gear for Long-Haul Transport down the Axon.

Accurate control of polarized cargo trafficking is essential for neuronal function. In this issue of Neuron, Gumy et al. (2017) show that MAP2 defines a pre-axonal filtering zone and controls axonal cargo transport by influencing the activities of distinct kinesin motors.

The Sorting Receptor SorCS1 Regulates Trafficking of Neurexin and AMPA Receptors.

The formation, function, and plasticity of synapses require dynamic changes in synaptic receptor composition. Here, we identify the sorting receptor SorCS1 as a key regulator of synaptic receptor trafficking. Four independent proteomic analyses identify the synaptic adhesion molecule neurexin and the AMPA glutamate receptor (AMPAR) as major proteins sorted by SorCS1. SorCS1 localizes to early and recycling endosomes and regulates neurexin and AMPAR surface trafficking. Surface proteome analysis of SorCS1-deficient neurons shows decreased surface levels of these, and additional, receptors. Quantitative in vivo analysis of SorCS1-knockout synaptic proteomes identifies SorCS1 as a global trafficking regulator and reveals decreased levels of receptors regulating adhesion and neurotransmission, including neurexins and AMPARs. Consequently, glutamatergic transmission at SorCS1-deficient synapses is reduced due to impaired AMPAR surface expression. SORCS1 mutations have been associated with autism and Alzheimer disease, suggesting that perturbed receptor trafficking contributes to synaptic-composition and -function defects underlying synaptopathies.

RKIP inhibition in cervical cancer is associated with higher tumor aggressive behavior and resistance to cisplatin therapy.

Cervical cancer is one of the most common cancers in women worldwide, being high-risk group the HPV infected, the leading etiological factor. The raf kinase inhibitory protein (RKIP) has been associated with tumor progression and metastasis in several human neoplasms, however its role on cervical cancer is unclear. In the present study, 259 uterine cervix tissues, including cervicitis, cervical intraepithelial lesions and carcinomas, were analyzed for RKIP expression by immunohistochemistry. We found that RKIP expression was significantly decreased during malignant progression, being highly expressed in non-neoplastic tissues (54% of the samples; 73/135), and expressed at low levels in the cervix invasive carcinomas (∼15% (19/124). Following in vitro downregulation of RKIP, we observed a viability and proliferative advantage of RKIP-inhibited cells over time, which was associated with an altered cell cycle distribution and higher colony number in a colony formation assay. An in vitro wound healing assay showed that RKIP abrogation is associated with increased migratory capability. RKIP downregulation was also associated with an increased vascularization of the tumors in vivo using a CAM assay. Furthermore, RKIP inhibition induced cervical cancer cells apoptotic resistance to cisplatin treatment. In conclusion, we described that RKIP protein is significantly depleted during the malignant progression of cervical tumors. Despite the lack of association with patient clinical outcome, we demonstrate, in vitro and in vivo, that loss of RKIP expression can be one of the factors that are behind the aggressiveness, malignant progression and chemotherapy resistance of cervical cancer.