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The logopenic variant of primary progressive aphasia (lvPPA) is the most recent variant of primary progressive aphasia (PPA) to be identified; thus far, it has been poorly investigated. Despite being typically associated with Alzheimer disease (AD), lvPPA has recently been linked to frontotemporal lobe degeneration (FTLD), with distinctive cognitive and neural features that are worthy of further investigation. Here, we describe the neuropsychological and linguistic profile, as well as cerebral abnormalities, of an individual exhibiting PPA and carrying a pathogenetic variant in the GRN gene, from a 3-year longitudinal perspective. The individual's initial profile resembled lvPPA because it was characterized by word-finding difficulties and phonological errors in spontaneous speech in addition to sentence repetition and phonological short-term memory impairments. The individual's structural and metabolic imaging data demonstrated left temporal and bilateral frontal atrophy and hypometabolism, respectively. On follow-up, as the pathology progressed, dysprosody, stereotypical speech patterns, agrammatism, and orofacial apraxia appeared, suggesting an overlap with the nonfluent variant of PPA (nfvPPA). Severe sentence comprehension impairment also became evident. Our longitudinal and multidisciplinary diagnostic approach allowed us to better characterize the progression of a GRN-positive lvPPA profile, providing neuropsychological and imaging indicators that might be helpful to improve classification between different PPA variants and to address a nosological issue. Finally, we discuss the importance of early diagnosis of PPA given the possible overlap between different PPA variants during the progression of the pathology.

Background and Hypothesis A primary disruption of the bodily self is considered a core feature of schizophrenia (SCZ). The “disembodied” self might be underpinned by inefficient body-related multisensory integration processes, normally occurring in the peripersonal space (PPS), a plastic sector of space surrounding the body whose extent is altered in SCZ. Although PPS is a malleable interface marking the perceptual border between self and others, no study has addressed the potential alteration of its plasticity in SCZ. We investigated the plasticity of PPS in SCZ patients after a motor training with a tool in the far space. Study Design Twenty-seven SCZ patients and 32 healthy controls (HC) underwent an audio-tactile task to estimate PPS boundary before (Session 1) and after (Session 3) the tool-use. Parameters of PPS, including the size and the slope of the psychometric function describing audio-tactile RTs as a function of the audio-tactile distances, were estimated. Study Results Results confirm a narrow PPS extent in SCZ. Surprisingly, we found PPS expansion in both groups, thus showing for the first time a preserved PPS plasticity in SCZ. Patients experienced a weaker differentiation from others, as indicated by a shallower PPS slope at Session 1 that correlated positively with negative symptoms. However, at Session 3, patients marked their bodily boundary in a steeper way, suggesting a sharper demarcation of PPS boundaries after the action with the tool. Conclusions These findings highlight the importance of investigating the multisensory and motor roots of self-disorders, paving the way for future body-centred rehabilitation interventions that could improve patients’ altered body boundary.

Background Several people affected by COVID-19 experienced neurological manifestations, altered sleep quality, mood disorders, and disability following hospitalization for a long time. Objective To explore the impact of different neurological symptoms on sleep quality, mood, and disability in a consecutive series of patients previously hospitalized for COVID-19 disease. Methods We evaluated 83 patients with COVID-19 around 3 months after hospital discharge. They were divided into 3 groups according to their neurological involvement (i.e., mild, unspecific, or no neurological involvement). Socio-demographic, clinical data, disability level, emotional distress, and sleep quality were collected and compared between the three groups. Results We found that higher disability, depressive symptoms, and lower sleep quality in patients with mild neurological involvement compared to patients with unspecific and no neurological involvement. Differences between groups were also found for clinical variables related to COVID-19 severity. Conclusion After 3 months from hospital discharge, patients with more severe COVID-19 and mild neurological involvement experienced more psychosocial alterations than patients with unspecific or no neurological involvement. Both COVID-19 and neurological manifestations’ severity should be considered in the clinical settings to plain tailored interventions for patients recovering from COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s10072-022-05907-1.

It is now accepted that reactive oxygen species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS‐controlled production is played by the NADPH oxidases (NOXs), a group of seven membrane‐bound enzymes (NOX1‐5 and DUOX1‐2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family member present in cyanobacteria, protists, plants, fungi, and the animal kingdom. We show that the calmodulin‐like regulatory EF‐domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C‐terminal lobe of the EF‐domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase (DH) domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the DH domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded‐to‐folded transition of the EF‐domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation., The N‐terminal domain (EF) of NADPH oxidase 5 contains four EF‐hand motifs and is the main player in this enzyme activation. In the absence of calcium, EF‐hands 3 and 4 are unfolded and the domain remains separated from the catalytic dehydrogenase domain (DH). In the presence of calcium, EF‐hands 3 and 4 acquire a structure and bind to the DH domain, activating the electron flow and production of reactive oxygen species.

Background: The multi-disciplinary tumor board (MTB) is essential to quality cancer care and currently recommended to offer the best personalized clinical approach, but little has been published regarding MTBs in neuro-oncology (nMTBs). The aim of the present paper is to describe our nMTB, to evaluate its impact on clinical management decisions, and to assess the role of neuroradiologists. Methods: The retrospective evaluation of the cases discussed at our nMTB from March 2017 to March 2020. From the electronic records, we extracted epidemiological, clinical and other specific data of nMTB. From the radiological records, we calculated data relating to the number, time for revision, and other specifications of MRI re-evaluation. Statistical analysis was performed. Results: a total of 447 discussions were analyzed, representing 342 patients. The requests for case evaluations came from radiation oncologists (58.8%) and neurosurgeons (40.5%), and were mainly addressed to the neuroradiologist (73.8%). The most frequent questions were about the treatment’s changes (64.4%). The change in patient treatment was reported in 40.5% of cases, 76.8% of these were based on the neuroradiologic assessment. A total of 1514 MRI examinations were re-evaluated, employing approximately 67 h overall. The median of the MRI exams reviewed per patient was 3 (min–max 1–12). Conclusions: Our study supported that the multidisciplinary approach to patient care can be particularly effective in managing brain tumors. A review by an expert neuroradiologist impacts patient management in the context of nMTBs, but has costs in terms of the time and effort spent preparing for it.

This study compares the incidence of new-onset symptoms within 24 hours after enhanced magnetic resonance imaging (eMRI) with intravenous administration of gadodiamide or gadoterate meglumine compared with a control group undergoing unenhanced MRI (uMRI).A prospective cohort study (n = 1088 patients) was designed to assess the incidence of symptoms within 24 hours after administration of gadodiamide or gadoterate meglumine. The participants underwent a structured questionnaire by phone call before and 24 hours after the MRI scan to check for symptoms that were not present before the scan. The questionnaire included a list of active questions aimed to test the prevalence of symptoms that have been proposed in the debated definition of gadolinium deposition disease (GDD) and that we recorded in this study as GDD-like. In particular, the following symptoms and signs were tested: central torso pain, arm or leg pain, bone pain, headache, skin redness (any site of the body), fatigue, and mental confusion.Fisher exact test was used to test differences between groups with significance threshold set at P0.05.Within the 24 hours after the MRI scan, 8.3% of patients reported at least one new-onset symptom in the uMRI group versus 17.4% in the gadodiamide eMRI versus 17.8% in the gadoterate meglumine eMRI group. The difference between the eMRI and the uMRI group was statistically significant (P0.001 for gadodiamide and P0.001 for gadoterate meglumine). There was not a different incidence of symptoms between the gadodiamide and the gadoterate meglumine eMRI groups. For gadodiamide, fatigue (P0.05) and dizziness (P0.05) were symptoms significantly more frequent than uMRI group; for gadoterate meglumine, fatigue (P0.01), mental confusion (P0.01), and diarrhea (P0.01) were significantly more frequent than uMRI group.We found that the onset of new symptoms within 24 hours after exposure to gadolinium-based contrast agent was more frequent than after uMRI. Among GDD-like symptoms, fatigue and mental confusion were the most frequent symptoms reported after eMRI. The other GDD-like symptoms were not overreported after eMRI versus uMRI. Thus, these results are questioning the term GDD.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6-Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2R/AT1R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21. © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. ISSN:0947-6539 ISSN:1521-3765

Body representation (BR) refers to the mental representation of motor, sensory, emotional and semantic information about the physical body. This cognitive representation is used in our everyday life, continuously, even though most of the time we do not appreciate it consciously. In some cases, BR is vital to be able to communicate. A crucial feature of signed languages (SLs), for instance, is that body parts such as hands are used to communicate. Nevertheless, little is known about BR in SL: is the communicative function of the body overwriting the physical constraints? Here, we explored this question by comparing twelve British Sign Language (BSL) learners to seventeen tango dancers (body expertise but not for communication) and fourteen control subjects (no special body expertise). We administered the Body Esteem Scale (BES), the Hand Laterality Task (HLT) and the Mental Motor Chronometry (MMC). To control for visual imagery, we administered ad hoc control tasks. We did not identify parameters able to differentiate between SL users and the other groups, whereas the more implicit parameters distinguished clearly tango dancers from controls. Importantly, neither tasks on visual imagery nor the BES revealed differences. Our findings offer initial evidence that linguistic use of the body not necessarily influences the cognitive components we explored of body representation.

With many crucial roles in enzymatic aerobic metabolism, the concentration of the heme must be tightly regulated. The heme exporter Feline Leukemia Virus sub-group C Receptor 1a (FLVCR1a), an integral membrane protein with twelve transmembrane helices, is a key player in the maintenance of cellular heme homeostasis. It was first identified as the host receptor for the Feline Leukemia Virus sub-group C (FeLV-C), a retrovirus causing hematological abnormalities in cats and other felines. Mutations in the Flvcr1 were later identified in human patients affected by Posterior Column Ataxia and Retinitis Pigmentosa (PCARP) and Hereditary Sensory and Autonomic Neuropathies (HSANs). Despite being an essential component in heme balance, currently there is a lack in the understanding of its function at the molecular level, including the effect of disease-causing mutations on protein function and structure. Therefore, there is a need for protocols to achieve efficient recombinant production yielding milligram amounts of highly pure protein to be used for biochemical and structural studies. Here, we report the first FLVCR1a reliable protocol suitable for both antibody generation and structural characterisation.

We typically misestimate the dimensions of our body e.g., we perceive our fingers as shorter, and our torso as more elongated, than they actually are. It stands to reason that those metric biases may also extend to objects that we interact with, to facilitate attunement with the environment. To explore this hypothesis, we compared the metric representations of seven objects and the subjects' own hand using the Line Length Judgment task, in six experiments involving 152 healthy subjects. We evaluated the size estimation errors made for each target (hand or previously observed objects) by asking subjects to compare the vertical or horizontal dimension of a specific target against the length of a vertical or horizontal line. As expected, we showed that the hand is misperceived in its dimensions. Interestingly, we found that metric biases are also present for daily-life objects, such as a mobile phone and a coffee mug, and are not affected by familiarity with the objects. In contrast, objects that are less likely to be manipulated, either because they are potentially harmful or disgusting, were differently represented. Furthermore, the propensity to interact with an object, rated by an independent sample of subjects, best predicted the pattern of metric biases associated with that object. Taken together, these findings support the hypothesis that biases affecting the hand representation extend to objects that elicit action-oriented behavior, highlighting the importance of studying the body as integrated and active in the environment.

Highlights • Acute DOC patients with favourable outcome show preserved event-related electrodermal response. • Acute DOC patients showed reduced fALFF in the posterior cingulate cortex. • Event-related electrodermal activity correlated with the fALFFs in the PCC in the acute phase., An accurate prognosis on the outcome of brain-injured patients with disorders of consciousness (DOC) remains a significant challenge, especially in the acute stage. In this study, we applied a multiple-technique approach to provide accurate predictions on functional outcome after 6 months in 15 acute DOC patients. Electrophysiological correlates of implicit cognitive processing of verbal stimuli and data-driven voxel-wise resting-state fMRI signals, such as the fractional amplitude of low-frequency fluctuations (fALFF), were employed. Event-related electrodermal activity, an index of autonomic activation, was recorded in response to emotional words and pseudo-words at baseline (T0). On the same day, patients also underwent a resting-state fMRI scan. Six months later (T1), patients were classified as outcome-negative and outcome-positive using a standard functional outcome scale. We then revisited the baseline measures to test their predictive power for the functional outcome measured at T1. We found that only outcome-positive patients had an earlier, higher autonomic response for words compared to pseudo-words, a pattern similar to that of healthy awake controls. Furthermore, DOC patients showed reduced fALFF in the posterior cingulate cortex (PCC), a brain region that contributes to autonomic regulation and awareness. The event-related electrodermal marker of residual cognitive functioning was found to have a significant correlation with residual local neuronal activity in the PCC. We propose that a residual autonomic response to cognitively salient stimuli, together with a preserved resting-state activity in the PCC, can provide a useful prognostic index in acute DOC.

NADPH-oxidases (NOXs) purposefully produce reactive-oxygen-species (ROS) and are found in most kingdoms of life. The seven human NOXs are each characterized by a specific expression profile and a fine regulation to spatio-temporally tune ROS concentration in cells and tissues. One of the best known roles for NOXs is in host protection against pathogens but ROS themselves are important second messengers involved in tissue regeneration and the modulation of pathways that induce and sustain cell proliferation. As such, NOXs are attractive pharmacological targets in immunomodulation, fibrosis and cancer. We have studied an extensive number of available NOX inhibitors, with the specific aim to identify bona fide ligands versus ROS-scavenging molecules. Accordingly, we have established a comprehensive platform of biochemical and biophysical assays. Most of the investigated small molecules revealed ROS-scavenging and/or assay-interfering properties to various degrees. A few compounds, however, were also demonstrated to directly engage one or more NOX enzymes. Diphenylene iodonium was found to react with the NOXs’ flavin and heme prosthetic groups to form stable adducts. We also discovered that two compounds, VAS2870 and VAS3947, inhibit NOXs through the covalent alkylation of a cysteine residue. Importantly, the amino acid involved in covalent binding was found to reside in the dehydrogenase domain, where the nicotinamide ring of NADPH is bound. This work can serve as a springboard to guide further development of bona fide ligands with either agonistic or antagonistic properties toward NOXs., Graphical abstract Image 1, Highlights • NOX inhibition by twenty-four compounds was tested using multiple assays. • Most of the evaluated compounds displayed significant ROS scavenging and/or assay-interfering effects. • DPI is demonstrated to engage the heme and the flavin cofactors of NOXs. • VAS2870 and VAS3947 are covalent bona-fide NOX inhibitors. • A conserved active-site cysteine can be targeted for inhibition.

The sense of body ownership is a fundamental feature that refers to the ability to recognize our body as our own, allowing us to interact properly with the outside world. Usually, it is explored by means of the Rubber Hand Illusion (RHI) during which a dummy hand is incorporated in the mental representation of one's own body throughout a multisensory (visuo-tactile) integration mechanism. Particular attention has been paid to the neurofunctional counterparts of this mechanism highlighting the pivotal role of an occipito-parieto-frontal network involving the Ventral Premotor area (PMv). To date, the specific role of the PMv in generating the sense of ownership is still unknown. In this study, we aimed at exploring the role of PMv in generating and experiencing the RHI. Off-line repetitive Transcranial Magnetic Stimulation (rTMS) was applied to a group of 24 healthy participants whilst changes in proprioceptive judgment and self-reported illusion sensations were collected and analysed separately. The PMv was not directly implicated in generating the sense of ownership. Indeed, its inhibition affected the explicit detection of the visuo-tactile congruence without interfering with the illusion experience itself. We hypothesized that the conscious visuo-tactile congruence detection may be independent from the conscious illusion experience. Also, our results support the view that the RHI grounds on a complex interaction between bottom-up and top-down processes, as the visuo-tactile integration per se may be not sufficient to trigger the subjective illusion.

NADPH oxidases (NOXs) are membrane enzymes whose sole function is the generation of reactive oxygen species. Humans have seven NOX isoenzymes that feature distinct functions in immune response and cell signaling but share the same catalytic core comprising a FAD-binding dehydrogenase domain and a heme-binding transmembrane domain. We previously described a mutation that stabilizes the dehydrogenase domain of a prokaryotic homolog of human NOX5. The thermostable mutant exhibited a large 19 °C increase in the apparent melting temperature (app T

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

NADPH oxidases (NOXs) are integral membrane enzymes that produce reactive oxygen species. Humans have seven NOX enzymes that feature a very similar catalytic core but distinct regulatory mechanisms. The recent structural elucidation of the NOX catalytic domains has been a step forward in the field. NADPH, FAD, and two hemes form a linear array of redox cofactors that transfer electrons across to the two sides of the membrane. Oxygen is reduced through an unusual outer sphere mechanism that does not involve any covalent intermediate with the heme iron. Several recent studies have expanded the roles of NOXs in cell signaling, innate immune response, and cell proliferation including oncogenic transformation. This work reinforces NOX-generated ROS as powerful signaling molecules. A challenging question is to understand the specific mechanisms of enzyme regulation and to harness the growing insight on NOXs' structure and biochemistry to generate more powerful small-molecule modulators of NOX activities.

Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues., Highlights • Human DHAPAT is associated to peroxisomal membrane through the N-terminal region. • Recombinant human DHAPAT expressed and purified from P. pastoris cells is active. • Evidence of the in vitro reconstitution of DHAPAT/ADPS enzymatic complex.

GPCR subtypes possess distinct functional and pharmacological profiles, and thus development of subtype-selective ligands has immense therapeutic potential. This is especially the case for the angiotensin receptor subtypes AT1R and AT2R, where a functional negative control has been described and AT2R activation highlighted as an important cancer drug target. We describe a strategy to fine-tune ligand selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions. Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analogue that exerted 18,000-fold higher selectivity for AT2R versus AT1R was obtained. We show that this compound is a negative regulator of AT1R signaling since it is able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.

Neuroimaging tools could open a window on residual neurofunctional activity in the absence of detectable behavioural responses in patients with disorders of consciousness (DOC). Nevertheless, the literature on this topic is characterised by a large heterogeneity of paradigms and methodological approaches that can undermine the reproducibility of the results. To explicitly test whether task-related functional magnetic resonance imaging (fMRI) can be used to systematically detect neurofunctional differences between different classes of DOC, and whether these differences are related with a specific category of cognitive tasks (either active or passive), we meta-analyzed 22 neuroimaging studies published between 2005 and 2017 using the Activation Likelihood Estimate method. The results showed that: (1) active and passive tasks rely on well-segregated patterns of activations, (2) both unresponsive wakeful syndrome and patients in minimally conscious state activated a large portion of the dorsal-attentional network, (3) shared activations between patients fell mainly in the passive activation map (7492 voxels), while only 48 voxels fell in a subcortical region of the active-map. Our results suggest that DOCs can be described along a continuum&mdash, rather than as separated clinical categories&mdash, and characterised by a widespread dysfunction of brain networks rather than by the impairment of a well functionally anatomically defined one.

The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

As part of the innate immune system, the complement system recognises a wide range of non-self structures present on pathogens or altered self cells. Its activation elicits proteolytic cascades which eventually results in the cleavage of the C5 protein into two fragments, C5a and C5b. The small anaphylatoxin C5a induces a variety of biological responses upon binding to the 7TM receptors C5aR and the C5L2, while the large C5b fragment nucleates formation of the membrane attack complex capable of killing susceptible pathogens by the formation of a pore structure in association with complement components C6, C7, C8, and C9. A number of regulatory molecules help to control C5 mediated immune responses towards host cells, but in several major inflammatory conditions including sepsis and arthritis, C5a is believed to contribute significantly to disease etiology. Inhibition of membrane attack complex assembly is already approved for treatment of paroxysmal nocturnal haemoglobinuria and atypical hemolytic uremic syndrome. A number of recent crystal structures have provided a comprehensive insight into the architecture and properties of intact C5 and its fragments, and how pathogens interfere with their function. Here we review the functional and structural aspects of C5 and its fragments, the pathological conditions associated with them, and strategies employed by pathogens to interfere with the biological function of C5. Structural insight and elucidation of evasion strategies employed by pathogens present a unique opportunity for promoting the development of novel selective C5 inhibitors with therapeutic applications.

Summary Methylxanthines, including caffeine and theophylline, are among the most widely consumed stimulant drugs in the world. These effects are mediated primarily via blockade of adenosine receptors. Xanthine analogs with improved properties have been developed as potential treatments for diseases such as Parkinson's disease. Here we report the structures of a thermostabilized adenosine A 2A receptor in complex with the xanthines xanthine amine congener and caffeine, as well as the A 2A selective inverse agonist ZM241385. The receptor is crystallized in the inactive state conformation as defined by the presence of a salt bridge known as the ionic lock. The complete third intracellular loop, responsible for G protein coupling, is visible consisting of extended helices 5 and 6. The structures provide new insight into the features that define the ligand binding pocket of the adenosine receptor for ligands of diverse chemotypes as well as the cytoplasmic regions that interact with signal transduction proteins.

Structural studies on mammalian integral membrane proteins have long been hampered by their instability in detergent. This is particularly true for the agonist conformation of G protein-coupled receptors (GPCRs), where it is thought that the movement of helices that occurs upon agonist binding results in a looser and less stable packing in the protein. Here, we show that mutagenesis coupled to a specific selection strategy can be used to stabilize the agonist and antagonist conformations of the adenosine A 2a receptor. Of the 27 mutations identified that improve the thermostability of the agonist conformation, only three are also present in the 17 mutations identified that improve the thermostability of the antagonist conformation, suggesting that the selection strategies used were specific for each conformation. Combination of the stabilizing mutations for the antagonist- or agonist-binding conformations resulted in mutants that are more stable at higher temperatures than the wild-type receptor by 17°C and 9°C, respectively. The mutant receptors both showed markedly improved stability in short-chain alkyl-glucoside detergents compared with the wild-type receptor, which will facilitate their structural analysis.

The serotonin transporter (SERT) is an integral membrane protein responsible for the clearance of serotonin from the synaptic cleft following the release of the neurotransmitter. SERT plays a prominent role in the regulation of serotoninergic neurotransmission and is a molecular target for multiple antidepressants as well as substances of abuse. Here we show that SERT associates with lipid rafts in both heterologous expression systems and rat brain and that the inclusion of the transporter into lipid microdomains is critical for serotonin uptake activity. SERT is present in a subpopulation of lipid rafts, which is soluble in Triton X-100 but insoluble in other non-ionic detergents such as Brij 58. Disaggregation of lipid rafts upon depletion of cellular cholesterol results in a decrease of serotonin transport capacity (V(max)), due to the reduction of turnover number of serotonin transport. Our data suggest that the association of SERT with lipid rafts may represent a mechanism for regulating the transporter activity and, consequently, serotoninergic signaling in the central nervous system, through the modulation of the cholesterol content in the cell membrane. Furthermore, SERT-containing rafts are detected in both intracellular and cell surface fractions, suggesting that raft association may be important for trafficking and targeting of SERT.

The serotonin transporter (SERT) plays a critical role in the maintenance of normal neurotransmission by serotonin [5-hydroxytryptamine (5-HT)]. Recent evidence suggests that SERT and other neurotransmitter transporters are tightly regulated. Activation of protein kinase C results in a decrease in SERT-mediated 5-HT uptake, which is due to an internalization of the transporter. However, to date little is known about the mechanism and proteins involved in the down-regulation of the transporter. One candidate SERT-regulatory protein is the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) protein, syntaxin 1A (Syn1A), which has recently been implicated in the regulation of ion channels as well as the SERT-related γ-aminobutyric acid- and glycine-transporters. Using 5-HT uptake assays, confocal microscopy and glutathione S-transferase (GST) pull-down assays we showed that Syn1A also interacts with SERT and alters the subcellular localization of the transporter, resulting in a reduction of 5-HT transport. In addition, we have used the yeast two-hybrid system to search for novel regulatory proteins that interact with the cytoplasmic N-terminal domain of SERT. By screening rat brain cDNA library we have identified six potential SERT-binding proteins. Here we also present progress towards the elucidation of the biological relevance of these proteins and their potential role for the regulation of the serotonin transporter.

Matteo Chiappedi,1 Alessio Toraldo,2 Silvia Mandrini,3 Federica Scarpina,2 Melissa Aquino,2 Francesca Giulia Magnani,2 Maurizio Bejor31Don Carlo Gnocchi ONLUS Foundation, Milan, Italy; 2University of Pavia, Department of Psychology, Pavia, Italy; 3University of Pavia, Department of Surgical, Resuscitative, Rehabilitative and Transplant Sciences, Pavia, ItalyIntroduction: Visual-motor skills are the basis for a great number of daily activities. To define a correct rehabilitation program for neurological patients who have impairment in these skills, there is a need for simple and cost-effective tools to determine which of the visual-motor system levels of organization are compromised by neurological lesions. In their 1995 book, The Visual Brain in Action (Oxford: Oxford University Press), AD Milner and MA Goodale proposed the existence of two pathways for the processing of visual information, the “ventral stream” and “dorsal stream,” that interact in movement planning and programming. Beginning with this model, our study aimed to validate a method to quantify the role of the ventral and dorsal streams in perceptual and visual-motor skills.Subjects and methods: Nineteen right-handed healthy subjects (mean age 22.8 years ± 3.18) with normal or corrected-to-normal vision were recruited. We proposed that a delayed pointing task, a distance reproduction task, and a delayed anti-pointing task could be used to assess the ventral stream, while the dorsal stream could be evaluated with a grasping task and an immediate pointing task. Performance was recorded and processed with the video-analysis software Dartfish ProSuite.Results: Results showed the expected pattern of predominance of attention for the superior left visual field, predominance of the flexor tone in proximal peri-personal space arm movements, tendency toward overestimation of short distances, and underestimation of long distances.Conclusion: We believe that our method is advantageous as it is simple and easily transported, but needs further testing in neurologically compromised patients.Keywords: dorsal stream, ventral stream, visual-motor skills, rehabilitation, neurological disorders

Structural studies on G-protein-coupled receptors have been hampered for many years by their instability in detergent solution and by the number of potential conformations that receptors can adopt. Recently, the structures of the beta(1) and beta(2) adrenergic receptors and the adenosine A(2a) receptor were determined in the antagonist-bound state, a receptor conformation that is thought to be more stable than the agonist-bound state. In contrast to these receptors, the neurotensin (NT) receptor NTS1 is much less stable in detergent solution. We have therefore used a systematic mutational approach coupled with activity assays to identify receptor mutants suitable for crystallization, both alone and in complex with the peptide agonist NT. The best receptor mutant NTS1-7m contained four point mutations. It showed increased stability compared to the wild-type receptor, in the absence of ligand, after solubilization with a variety of detergents. In addition, NTS1-7m bound to NT was more stable than unliganded NTS1-7m. Of the four thermostabilizing mutations, only one residue (A86L) is predicted to be in the lipid environment. In contrast, I260A appears to be buried within the transmembrane helix bundle, F342A may form a distant part of the putative ligand-binding site, whereas F358A is likely to be in a region that is important for receptor activation. NTS1-7m binds NT with a similar affinity for the wild-type receptor. However, agonist dissociation was slower, and NTS1-7m activated G-proteins poorly. The affinity of NTS1-7m for the antagonist SR48692 was also lower than that of the wild-type receptor. Thus, we have successfully stabilized NTS1 in an agonist-binding conformation that does not efficiently couple to G-proteins.

There are ≈350 non-odorant G protein-coupled receptors (GPCRs) encoded by the human genome, many of which are predicted to be potential therapeutic targets, but there are only two structures available to represent the whole of the family. We hypothesized that improving the detergent stability of these receptors and simultaneously locking them into one preferred conformation will greatly improve the chances of crystallization. We developed a generic strategy for the isolation of detergent-solubilized thermostable mutants of a GPCR, the β1-adrenergic receptor. The most stable mutant receptor, βAR-m23, contained six point mutations that led to an apparent T m 21°C higher than the native protein, and, in the presence of bound antagonist, βAR-m23 was as stable as bovine rhodopsin. In addition, βAR-m23 was significantly more stable in a wide range of detergents ideal for crystallization and was preferentially in an antagonist conformation in the absence of ligand.

The serotonin transporter (SERT) plays a critical role in the maintenance of normal neurotransmission by serotonin [5-hydroxytryptamine (5-HT)]. Recent evidence suggests that SERT and other neurotransmitter transporters are tightly regulated. Activation of protein kinase C results in a decrease in SERT-mediated 5-HT uptake, which is due to an internalization of the transporter. However, to date little is known about the mechanism and proteins involved in the down-regulation of the transporter. One candidate SERT-regulatory protein is the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) protein, syntaxin 1A (Syn1A), which has recently been implicated in the regulation of ion channels as well as the SERT-related gamma-aminobutyric acid- and glycine-transporters. Using 5-HT uptake assays, confocal microscopy and glutathione S-transferase (GST) pull-down assays we showed that Syn1A also interacts with SERT and alters the subcellular localization of the transporter, resulting in a reduction of 5-HT transport. In addition, we have used the yeast two-hybrid system to search for novel regulatory proteins that interact with the cytoplasmic N-terminal domain of SERT. By screening rat brain cDNA library we have identified six potential SERT-binding proteins. Here we also present progress towards the elucidation of the biological relevance of these proteins and their potential role for the regulation of the serotonin transporter.

Several reports have described a role of macrophagic, endothelial and synoviocytal nitric oxide (NO) in inflammation, immunity and sensory processes in joint diseases. In view of the role of the peripheral nervous system in arthritis and owing to the presence of NO-producing neurons in primary sensory neurons, we have investigated the possible role of neuronal NO during adjuvant-induced joint inflammation in rats. Neural nitric oxide synthase production in sensory ganglia and the spinal cord was investigated by in situ hybridization and immunocytochemistry. Neuronal NO synthase mRNA expression and neuronal NO synthase immunoreactivity increased in lumbar dorsal root ganglia in arthritic rats compared to those of normal rats, whereas neuronal NO synthase mRNA expression decreased in lamina X and lamina I-II of the lumbar spinal cord. The administration of the selective neuronal NO synthase inhibitor 7-nitro indazole, reduced the joint inflammation, whereas the administration of the inducible NO synthase selective inhibitor, aminoguanidine, had no effect on inflammation when administered daily from the third day after adjuvant. These findings could indicate a role for neural NO in adjuvant arthritis.