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.

The Ubiquitinated Axon: Local Control of Axon Development and Function by Ubiquitin.

Ubiquitin tagging sets protein fate. With a wide range of possible patterns and reversibility, ubiquitination can assume many shapes to meet specific demands of a particular cell across time and space. In neurons, unique cells with functionally distinct axons and dendrites harboring dynamic synapses, the ubiquitin code is exploited at the height of its power. Indeed, wide expression of ubiquitination and proteasome machinery at synapses, a diverse brain ubiquitome, and the existence of ubiquitin-related neurodevelopmental diseases support a fundamental role of ubiquitin signaling in the developing and mature brain. While special attention has been given to dendritic ubiquitin-dependent control, how axonal biology is governed by this small but versatile molecule has been considerably less discussed. Herein, we set out to explore the ubiquitin-mediated spatiotemporal control of an axon's lifetime: from its differentiation and growth through presynaptic formation, function, and pruning.

Botulinum toxin as a novel therapeutic approach for auriculotemporal neuralgia.

OBJECTIVES/BACKGROUND: Auriculotemporal neuralgia is a rare headache disorder. Anesthetic nerve blockade seems to be effective in most cases; however, literature is scarce about the management of refractory cases. METHODS: Case report. RESULTS: We report a 44-year-old woman with migraine without aura presented with moderate-to-severe right temporoparietal headache. The pain was refractory to multiple pharmacological strategies, including intravenous analgesia. A more throughout examination lead to the diagnosis of auriculotemporal neuralgia and anesthetic nerve blocks were performed. Due to early pain recrudescence, botulinum toxin (BoNT) was tried with better and longstanding pain control. CONCLUSION: BoNT may be a useful treatment option in refractory auriculotemporal neuralgia. The best approach is yet to be established; however, the "follow-the-pain" protocol may be a reasonable option.

Machine Learning Approach for Fatigue Estimation in Sit-to-Stand Exercise.

Physical exercise (PE) has become an essential tool for different rehabilitation programs. High-intensity exercises (HIEs) have been demonstrated to provide better results in general health conditions, compared with low and moderate-intensity exercises. In this context, monitoring of a patients' condition is essential to avoid extreme fatigue conditions, which may cause physical and physiological complications. Different methods have been proposed for fatigue estimation, such as: monitoring the subject's physiological parameters and subjective scales. However, there is still a need for practical procedures that provide an objective estimation, especially for HIEs. In this work, considering that the sit-to-stand (STS) exercise is one of the most implemented in physical rehabilitation, a computational model for estimating fatigue during this exercise is proposed. A study with 60 healthy volunteers was carried out to obtain a data set to develop and evaluate the proposed model. According to the literature, this model estimates three fatigue conditions (low, moderate, and high) by monitoring 32 STS kinematic features and the heart rate from a set of ambulatory sensors (Kinect and Zephyr sensors). Results show that a random forest model composed of 60 sub-classifiers presented an accuracy of 82.5% in the classification task. Moreover, results suggest that the movement of the upper body part is the most relevant feature for fatigue estimation. Movements of the lower body and the heart rate also contribute to essential information for identifying the fatigue condition. This work presents a promising tool for physical rehabilitation.

Puzzling out presynaptic differentiation.

Proper brain function in the nervous system relies on the accurate establishment of synaptic contacts during development. Countless synapses populate the adult brain in an orderly fashion. In each synapse, a presynaptic terminal loaded with neurotransmitters-containing synaptic vesicles is perfectly aligned to an array of receptors in the postsynaptic membrane. Presynaptic differentiation, which encompasses the events underlying assembly of new presynaptic units, has seen notable advances in recent years. It is now consensual that as a growing axon encounters the receptive dendrites of its partner, presynaptic assembly will be triggered and specified by multiple postsynaptically-derived factors including soluble molecules and cell adhesion complexes. Presynaptic material that reaches these distant sites by axonal transport in the form of pre-assembled packets will be retained and clustered, ultimately giving rise to a presynaptic bouton. This review focuses on the cellular and molecular aspects of presynaptic differentiation in the central nervous system, with a particular emphasis on the identity of the instructive factors and the intracellular processes used by neuronal cells to assemble functional presynaptic terminals. We provide a detailed description of the mechanisms leading to the formation of new presynaptic terminals. In brief, soma-derived packets of pre-assembled material are trafficked to distant axonal sites. Synaptogenic factors from dendritic or glial provenance activate downstream intra-axonal mediators to trigger clustering of passing material and their correct organization into a new presynaptic bouton. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

Diabetes induces changes in KIF1A, KIF5B and dynein distribution in the rat retina: implications for axonal transport.

Diabetic retinopathy is a leading cause of vision loss and blindness. Disruption of axonal transport is associated with many neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated the impact of type 1 diabetes (2 and 8 weeks duration) on KIF1A, KIF5B and dynein motor proteins in the retina. Additionally, since hyperglycemia is considered the main trigger of diabetic complications, we investigated whether prolonged exposure to elevated glucose could affect the content and distribution of motor proteins in retinal cultures. The immunoreactivity of motor proteins was evaluated by immunohistochemistry in retinal sections and by immunoblotting in total retinal extracts from streptozotocin-induced diabetic and age-matched control animals. Primary retinal cultures were exposed to high glucose (30 mM) or mannitol (osmotic control; 24.5 mM plus 5.5 mM glucose), for seven days. Diabetes decreased the content of KIF1A at 8 weeks of diabetes as well as KIF1A immunoreactivity in the majority of retinal layers, except for the photoreceptor and outer nuclear layer. Changes in KIF5B immunoreactivity were also detected by immunohistochemistry in the retina at 8 weeks of diabetes, being increased at the photoreceptor and outer nuclear layer, and decreased in the ganglion cell layer. Regarding dynein immunoreactivity there was an increase in the ganglion cell layer after 8 weeks of diabetes. No changes were detected in retinal cultures. These alterations suggest that axonal transport may be impaired under diabetes, which might contribute to early signs of neural dysfunction in the retina of diabetic patients and animal models.

Excitotoxicity downregulates TrkB.FL signaling and upregulates the neuroprotective truncated TrkB receptors in cultured hippocampal and striatal neurons.

Brain-derived neurotrophic factor (BDNF) plays an important role in neuronal survival through activation of TrkB receptors. The trkB gene encodes a full-length receptor tyrosine kinase (TrkB.FL) and its truncated (T1/T2) isoforms. We investigated the changes in TrkB protein levels and signaling activity under excitotoxic conditions, which are characteristic of brain ischemia, traumatic brain injury, and neurodegenerative disorders. Excitotoxic stimulation of cultured rat hippocampal or striatal neurons downregulated TrkB.FL and upregulated a truncated form of the receptor (TrkB.T). Downregulation of TrkB.FL was mediated by calpains, whereas the increase in TrkB.T protein levels required transcription and translation activities. Downregulation of TrkB.FL receptors in hippocampal neurons correlated with a decrease in BDNF-induced activation of the Ras/ERK and PLCγ pathways. However, calpain inhibition, which prevents TrkB.FL degradation, did not preclude the decrease in signaling activity of these receptors. On the other hand, incubation with anisomycin, to prevent the upregulation of TrkB.T, protected to a large extent the TrkB.FL signaling activity, suggesting that truncated receptors may act as dominant-negatives. The upregulation of TrkB.T under excitotoxic conditions was correlated with an increase in BDNF-induced inhibition of RhoA, a mediator of excitotoxic neuronal death. BDNF fully protected hippocampal neurons transduced with TrkB.T when present during excitotoxic stimulation with glutamate, in contrast with the partial protection observed in cells overexpressing TrkB.FL or expressing GFP. These results indicate that BDNF protects hippocampal neurons by two distinct mechanisms: through the neurotrophic effects of TrkB.FL receptors and by activation of TrkB.T receptors coupled to inhibition of the excitotoxic signaling.

IL-4 shapes microglia-dependent pruning of the cerebellum during postnatal development.

Interleukin-4 (IL-4) is a type 2 cytokine with pleiotropic functions in adaptive immunity, allergies, and cognitive processes. Here, we show that low levels of IL-4 in the early postnatal stage delineate a critical period in which microglia extensively prune cerebellar neurons. Elevating the levels of this cytokine via peripheral injection, or using a mouse model of allergic asthma, leads to defective pruning, permanent increase in cerebellar granule cells, and circuit alterations. These animals also show a hyperkinetic and impulsive-like phenotype, reminiscent of attention-deficit hyperactivity disorder (ADHD). These alterations are blocked in Il4rαfl/fl::Cx3cr1-CreER mice, which are deficient in IL-4 receptor signaling in microglia. These findings demonstrate a previously unknown role for IL-4 during a neuroimmune critical period of cerebellar maturation and provide a first putative mechanism for the comorbidity between allergic disease and ADHD observed in humans.

Insulin treatment may not be associated with worse prognosis in acute heart failure diabetic patients.

BACKGROUND: Diabetic patients with heart failure (HF) treated with insulin appear to have a worse prognosis compared to oral anti-diabetic (OAD) agents. Whether insulin is a risk factor or a marker of disease severity is unknown. We studied the prognostic impact of insulin treatment in an acute HF diabetic population. METHODS: From a prospectively recruited population of hospital-admitted acute HF patients we retrospectively selected a convenience sample. Pair-matched analysis: each insulin-treated patient was matched with a diabetic patient with similar glycated hemoglobin (HbA1c±0.2%) under OAD. End-point: all-cause death. FOLLOW-UP: 5 years. Insulin-treated and OAD-treated patients were compared. Multivariate Cox-regression analysis was used to analyze the prognostic impact of insulin. RESULTS: We studied 92 patients: 46 insulin-treated and 46 HbA1c-matched under OAD. Mean age: 74±9 years, 46.7% male and 63.5% had HF with reduced ejection fraction. HbA1c was 7.8±1.5% in both groups. In the subgroup under OAD: 87.0% metformin, 41.3% sulphonylurea, 28.3% dipeptidyl peptidase-4 inhibitors and 4.3% other agents. Insulin-treated patients had lower hemoglobin, higher creatinine and discharged B-type natriuretic peptide (BNP) levels (816.6 [289.2-1805.8] vs. 613.3 [205.6-1110.8]; P=0.02). Seventy three patients died. There were no differences in mortality up to 5 years. After multivariate adjustment accounting for hemoglobin, creatinine and discharge BNP, HR for 1-, 2-, 3-, and 5-year all-cause death in insulin-treated patients were 1.48 (0.62-3.54); P=0.38, 1.11 (0.55-2.25); P=0.77, 1.08 (0.56-2.08); P=0.28 and 1.24 (0.70-2.19); P=0.46, respectively. CONCLUSIONS: Insulin-treated diabetic patients with HF and HbA1c-matched patients treated with OAD have similarly ominous prognosis. Our results favor insulin as a marker of poor health condition.

Visualizing K48 Ubiquitination during Presynaptic Formation By Ubiquitination-Induced Fluorescence Complementation (UiFC).

In recent years, signaling through ubiquitin has been shown to be of great importance for normal brain development. Indeed, fluctuations in ubiquitin levels and spontaneous mutations in (de)ubiquitination enzymes greatly perturb synapse formation and neuronal transmission. In the brain, expression of lysine (K) 48-linked ubiquitin chains is higher at a developmental stage coincident with synaptogenesis. Nevertheless, no studies have so far delved into the involvement of this type of polyubiquitin chains in synapse formation. We have recently proposed a role for polyubiquitinated conjugates as triggering signals for presynaptic assembly. Herein, we aimed at characterizing the axonal distribution of K48 polyubiquitin and its dynamics throughout the course of presynaptic formation. To accomplish so, we used an ubiquitination-induced fluorescence complementation (UiFC) strategy for the visualization of K48 polyubiquitin in live hippocampal neurons. We first validated its use in neurons by analyzing changing levels of polyubiquitin. UiFC signal is diffusely distributed with distinct aggregates in somas, dendrites and axons, which perfectly colocalize with staining for a K48-specific antibody. Axonal UiFC aggregates are relatively stable and new aggregates are formed as an axon grows. Approximately 65% of UiFC aggregates colocalize with synaptic vesicle clusters and they preferentially appear in the axonal domains of axo-somatodendritic synapses when compared to isolated axons. We then evaluated axonal accumulation of K48 ubiquitinated signals in bead-induced synapses. We observed rapid accumulation of UiFC signal and endogenous K48 ubiquitin at the sites of newly formed presynapses. Lastly, we show by means of a microfluidic platform, for the isolation of axons, that presynaptic clustering on beads is dependent on E1-mediated ubiquitination at the axonal level. Altogether, these results indicate that enrichment of K48 polyubiquitin at the site of nascent presynaptic terminals is an important axon-intrinsic event for presynaptic differentiation.

The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates.

Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin-proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses.

Identification of H-type BSE in Portugal.

During the bovine spongiform encephalopathy (BSE) epidemic, Portugal was the third most affected country. As a result of a successful national eradication plan, the number of BSE affected animals has been progressively declining in Portugal with no cases identified in 2013. However, within the scope of this active surveillance scheme, we have identified the first H-type BSE case born after the introduction of the reinforced ban in fallen stock. Here, we report the phenotypic features of this case and the analysis of the protein coding sequence of prnp as well as the prnp promoter and intron 1 insertion-deletions.

Diabetes alters KIF1A and KIF5B motor proteins in the hippocampus.

Diabetes mellitus is the most common metabolic disorder in humans. Diabetic encephalopathy is characterized by cognitive and memory impairments, which have been associated with changes in the hippocampus, but the mechanisms underlying those impairments triggered by diabetes, are far from being elucidated. The disruption of axonal transport is associated with several neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated the effect of diabetes (2 and 8 weeks duration) on KIF1A, KIF5B and dynein motor proteins, which are important for axonal transport, in the hippocampus. The mRNA expression of motor proteins was assessed by qRT-PCR, and also their protein levels by immunohistochemistry in hippocampal slices and immunoblotting in total extracts of hippocampus from streptozotocin-induced diabetic and age-matched control animals. Diabetes increased the expression and immunoreactivity of KIF1A and KIF5B in the hippocampus, but no alterations in dynein were detected. Since hyperglycemia is considered a major player in diabetic complications, the effect of a prolonged exposure to high glucose on motor proteins, mitochondria and synaptic proteins in hippocampal neurons was also studied, giving particular attention to changes in axons. Hippocampal cell cultures were exposed to high glucose (50 mM) or mannitol (osmotic control; 25 mM plus 25 mM glucose) for 7 days. In hippocampal cultures incubated with high glucose no changes were detected in the fluorescence intensity or number of accumulations related with mitochondria in the axons of hippocampal neurons. Nevertheless, high glucose increased the number of fluorescent accumulations of KIF1A and synaptotagmin-1 and decreased KIF5B, SNAP-25 and synaptophysin immunoreactivity specifically in axons of hippocampal neurons. These changes suggest that anterograde axonal transport mediated by these kinesins may be impaired in hippocampal neurons, which may lead to changes in synaptic proteins, thus contributing to changes in hippocampal neurotransmission and to cognitive and memory impairments.

an in vitro study comparing the intracanal effectiveness of calcium hydroxide- and linezolid-based medicaments against Enterococcus faecalis.

INTRODUCTION: This study evaluated the efficacy of calcium hydroxide (CH), Vitapex (VP), linezolid (LZ), a combination of LZ with CH (LC), and a control group (N, no medicament) against Enterococcus faecalis (EF). METHODS: Human single-rooted premolars were instrumented up to ProTaper size F3 files. EF suspension was inoculated into each root specimen and incubated. The medicaments were syringed into each root by weight and incubated. After 72 hours, 6 samples per group (among the 5 groups) were retrieved. A hole was drilled on each root, and the dentinal shavings obtained were allowed to fall in brain-heart infusion (BHI) broth. Dilutions from the broth were plated and spread over BHI agar and blood agar. Colony-forming units (CFU) of EF were measured from BHI agar. The procedure was repeated after 8 days and 14 days. RESULTS: In group CH, the mean CFU (log 10 values) after 72 hours, 8 days, and 14 days were 1.17 ± 1.16, 3.33 ± 1.97, and 4.17 ± 1.17, respectively (statistically significant). In group VP, the mean CFU were 0.83 ± 0.75, 4.00 ± 1.67, and 4.83 ± 1.72. In group LZ, the mean CFU at 72 hours and after 8 days was 0.17 ± 0.41, and no CFU were found on the fourteenth day. Similarly, in group LC, the mean CFU at 72 hours and after 8 days was 0.50 ± 0.84, which increased to 1.33 ± 1.51 on the fourteenth day (not significant). CONCLUSIONS: LZ was found to be most effective on EF, followed by LC, CH, and VP.