Galanin diminishes cortical spreading depolarization across rodents - A candidate for treatment?

Galanin (Gal) is a neuropeptide with the potential to ameliorate cortical spreading depolarization (CSD), an electrophysiological phenomenon occurring after brain injury or in migraine aura. Gal is expressed in all cortical neurons both in rat and in mouse cortices. Here we investigated whether the effect of Gal on CSD previously described in the rat is conserved in the mouse cortex. In rats, the topical application of Gal to the cortex for 1 h did not induce any change in CSD amplitudes, propagation velocity, or threshold of elicitation. Rather, topical application of Gal for 3 h was necessary to obtain a significant decrease in these CSD parameters and to develop a remarkable increase in the KCl threshold to elicit a CSD in rat cortex. In contrast, the topical application of Gal on cortical surface for 1 h in mice was sufficient to significantly attenuate CSD amplitudes and increase threshold. A thinner cortex, a faster diffusion or different affinity/expression of receptors for Gal are possible reasons to explain this difference in the time course between rats and mice. Our data are relevant to postulate Gal as a potential target for inhibition of CSD under pathological situations such as stroke or ischemia. SIGNIFICANCE STATEMENT: The neuropeptide Galanin (Gal) is expressed in all neurons throughout the cerebral cortex, both in rats and mice, and is able to reduce or even inhibit Cortical Spreading Depolarization, thus, Gal has the potential to control neuronal excitability that may identify Gal as a target in drug development against CSD.

Neuropathological changes in the TASTPM mouse model of Alzheimer's disease and their relation to hyperexcitability and cortical spreading depolarization.

Although Alzheimer's disease (AD) is characterized by distinct pathological changes, their precise impact on cortical functions are not well understood. Here we used TASTPM mice as an AD model and asked whether the development of neurodegenerative changes has an impact on the extracellular space (ECS) and neuronal excitability, in particular cortical spreading depolarization (CSD) which requires intact neuron and glial functions. We studied wildtype (WT) and TASTPM mice (3, 6, and 12 months old). TASTPM mice showed progressive proliferation of neocortical Amyloid-beta (Aß) plaques between 3 and 12 months (more deposits in females than in males) and Aß accumulation in cortical vessels. As plaques proliferated, neuroinflammatory microglial reaction (CD68, CD39 and Galectin-3) and astrogliosis (GFAP) developed progressively. The cortical ECS volume shrank significantly to about half the size of the WT. CSD in both WT and TASTPM mice showed considerable heterogeneity but did not correlate with the histological changes. However, CSDs were easier to elicit in TASTPM than in WT mice at 3 months, and also compared to older TASTPM mice. Moreover, TASTPM mice showed more hyperexcitability manifested as clonic-tonic behavior after sodium thiopental anesthesia. Thus, AD pathology was associated with abnormal hyperexcitability but did not homogenously alter CSD susceptibility.

Cortical spreading depolarization is a potential target for rat brain excitability modulation by Galanin.

The inhibitory neuropeptide Galanin (Gal) has been shown to mediate anticonvulsion and neuroprotection. Here we investigated whether Gal affects cortical spreading depolarization (CSD). CSD is considered the pathophysiological neuronal mechanism of migraine aura, and a neuronal mechanism aggravating brain damage upon afflictions of the brain. Immunohistochemistry localized Gal and the Gal receptors 1-3 (GalR1-3) in native rat cortex and evaluated microglial morphology after exposure to Gal. In anesthetized rats, Gal was applied alone and together with the GalR antagonists M40, M871, or SNAP 37889 locally to the exposed cortex. The spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection were measured. In rat cortex, Gal was present in all neurons of all cortical layers, but not in astrocytes, microglia and vessels. GalR2 and GalR3 were expressed throughout all neurons, whereas GalR1 was preponderantly located at neurons in layers IV and V, but only in about half of the neurons. In susceptible rats, topical application of Gal on cortex decreased CSD amplitude, slowed CSD propagation velocity, and increased the threshold for KCl to ignite CSD. In some rats, washout of previously applied Gal induced periods of epileptiform patterns in the electrocorticogram. Blockade of GalR2 by M871 robustly prevented all Gal effects on CSD, whereas blockade of GalR1 or GalR3 was less effective. Although microglia did not express GalRs, topical application of Gal changed microglial morphology indicating microglial activation. This effect of Gal on microglia was prevented by blocking neuronal GalR2. In conclusion, Gal has the potential to ameliorate CSD thus reducing pathophysiological neuronal events caused by or associated with CSD.

Recycling of polystyrene-based external thermal insulation composite systems - Application of combined mechanical and chemical recycling.

The material recycling of complex waste streams such as external thermal insulation composite systems (ETICS) is challenging, which is why their recycling in the sense of a circular economy is currently hardly established. Therefore, the combined mechanical and thermochemical recycling of ETICS based on expanded polystyrene (EPS) is investigated experimentally and by simulating full process chains in order to evaluate circular economy opportunities. Model ETICS as example for building and construction waste is pretreated mechanically, followed by either pyrolysis and / or gasification steps, and full mass and energy balances are derived. By the combined recycling, inorganic compounds can be separated to a large extent allowing a pre-concentrate generation. The plastic-rich pre-concentrate is converted into either pyrolysis oil with a high styrene monomer content of 51 wt% or to synthesis gas in the subsequent thermochemical conversions. The holistic approach enables a high carbon recycling rate between 53 and 68 wt%. In addition, the investigation reveals technology limitations and opportunities to be further developed and optimized.

From spreading depolarization to epilepsy with neuroinflammation: The role of CGRP in cortex.

CGRP release plays a major role in migraine pain by activating the trigeminal pain pathways. Here we explored putative additional effects of CGRP on cortical circuits and investigated whether CGRP affects cortical excitability, cortical spreading depolarization (CSD), a phenomenon associated with migraine aura, blood-brain-barrier (BBB) and microglial morphology. We used immunohistochemistry to localize CGRP and the CGRP receptor (CGRP-R) in native cortex and evaluated morphology of microglia and integrity of the BBB after exposure to CGRP. In anesthetized rats we applied CGRP and the CGRP-R antagonist BIBN4096BS locally to the exposed cortex and monitored the spontaneous electrocorticogram and CSDs evoked by remote KCl pressure microinjection. In mouse brain slices CGRP effects on neuronal activity were explored by multielectrode array. CGRP immunoreactivity was detectable in intracortical vessels, and all cortical neurons showed CGRP-R immunoreactivity. In rat cortex in vivo, topical CGRP induced periods of epileptiform discharges, however, also dose-dependently reduced CSD amplitudes and propagation velocity. BIBN4096BS prevented these effects. CGRP evoked synchronized bursting activity in mouse cortical but not in cerebellar slices. Topical application of CGRP to rat cortex induced plasma extravasation and this was associated with reduced ramification of microglial cells. From these findings we conclude that CGRP induces a pathophysiological state in the cortex, consisting in neuronal hyperexcitability and neuroinflammation. Thus, CGRP may have a pronounced impact on brain functions during migraine episodes supporting the benefit of CGRP antagonists for clinical use. However, increased cortical CGRP may end the CSD-induced aura phase of migraine.

Population genetics and plant growth experiments as prerequisite for conservation measures of the rare European aquatic plant Luronium natans (Alismataceae).

Information provided by population genetic studies is often necessary to effectively protect endangered species. In general, such data is scarce for aquatic plants and this holds also for Luronium natans, an aquatic macrophyte endemic to northwestern and western Europe. It is threatened across its whole distribution range due to human influences, in particular due to eutrophication and intensive fish farming. In spite of habitat protection populations continue to decline and re-introductions are one possibility to prevent the species' extinction. Therefore, insights in genetic diversity and relatedness of source populations is warranted. Thus, we performed Amplified Fragment-Length Polymorphism (AFLP) on two large populations in Saxony, Germany (Großenhainer Pflege and Niederspree), complemented with numerous additional occurrences from Europe. In addition, we conducted experiments on plant growth to assess optimal conditions for ex-situ cultivation taking water temperature, water level and substrate into account. We revealed considerably high levels of genetic diversity within populations (Shannon Indices ranged from 0.367 to 0.416) implying that populations are not restricted to clonal growth only but reproduce also by open-pollinated flowers. Remarkably, the two geographically close Saxon populations were genetically distant to each other but subpopulations within a locality were completely intermingled. Concerning optimal cultivation conditions, longest roots were obtained at temperatures >14°C and saturated, but not submerging water levels. Thus, our findings advocate for a re-introduction scheme from nearby source populations and provide detailed information on successful ex-situ cultivation.

Transient Receptor Potential vanilloid 4 ion channel in C-fibres is involved in mechanonociception of the normal and inflamed joint.

The Transient Receptor Potential vanilloid 4 ion channel (TRPV4) is an important sensor for osmotic and mechanical stimuli in the musculoskeletal system, and it is also involved in processes of nociception. In this study we investigated the putative role of TRPV4 ion channels in joint pain. In anesthetized rats we recorded from mechanosensitive nociceptive A∂- and C-fibres supplying the medial aspect of the knee joint. The intraarticular injection of the TRPV4 antagonist RN-1734 into the knee joint reduced the responses of C-fibres of the normal joint to noxious mechanical stimulation and the responses of the sensitized C-fibres of the acutely inflamed joint to innocuous and noxious mechanical stimulation. The responses of nociceptive A∂-fibres were not significantly altered by RN-1734. The intraarticular application of the TRPV4 agonists 4αPDD, GSK 1016790 A, and RN-1747 did not consistently alter the responses of A∂- and C-fibres to mechanical stimulation of the joint nor did they induce ongoing activity. We conclude that TRPV4 ion channels are involved in the responses of C-fibres to noxious mechanical stimulation of the normal joint, and in the enhanced sensitivity of C-fibres to mechanical stimulation of the joint during inflammation of the joint.

Crystal structure of idelalisib tert-butanol monosolvate dihydrate.

In the title structure, 5-fluoro-3-phenyl-2-[(1S)-1-(9H-purin-6-yl-amino)-prop-yl]quinazolin-4(3H)-one (= idelalisib) tert-butanol monosolvate dihydrate, C22H18FN7O·C4H10O·2H2O, the idelalisib mol-ecule displays planar quinazoline and purine systems which are nearly perpendicular to one another. Seven distinct hydrogen-bonding inter-actions link the idelalisib, t-BuOH and water mol-ecules into a complex chain structure with the topology of a 2,3,4,5-connected 4-nodal net having the point symbol (3.4.52.62)(3.4.52.64.72)(3.5.6)(5).

ROMO1 is a constituent of the human presequence translocase required for YME1L protease import.

The mitochondrial presequence translocation machinery (TIM23 complex) is conserved between the yeast Saccharomyces cerevisiae and humans; however, functional characterization has been mainly performed in yeast. Here, we define the constituents of the human TIM23 complex using mass spectrometry and identified ROMO1 as a new translocase constituent with an exceptionally short half-life. Analyses of a ROMO1 knockout cell line revealed aberrant inner membrane structure and altered processing of the GTPase OPA1. We show that in the absence of ROMO1, mitochondria lose the inner membrane YME1L protease, which participates in OPA1 processing and ROMO1 turnover. While ROMO1 is dispensable for general protein import along the presequence pathway, we show that it participates in the dynamics of TIM21 during respiratory chain biogenesis and is specifically required for import of YME1L. This selective import defect can be linked to charge distribution in the unusually long targeting sequence of YME1L. Our analyses establish an unexpected link between mitochondrial protein import and inner membrane protein quality control.

The potential of substance P to initiate and perpetuate cortical spreading depression (CSD) in rat in vivo.

The tachykinin substance P (SP) increases neuronal excitability, participates in homeostatic control, but induces brain oedema after stroke or trauma. We asked whether SP is able to induce cortical spreading depression (CSD) which often aggravates stroke-induced pathology. In anesthetized rats we applied SP (10-5, 10-6, 10-7, or 10-8 mol/L) to a restricted cortical area and recorded CSDs there and in remote non-treated areas using microelectrodes. SP was either applied in artificial cerebrospinal fluid (ACSF), or in aqua to perform a preconditioning. Plasma extravasation in cortical grey matter was assessed with Evans Blue. Only SP dissolved in aqua induced self-regenerating CSDs. SP dissolved in ACSF did not ignite CSDs even when excitability was increased by acetate-preconditioning. Aqua alone elicited as few CSDs as the lowest concentration of SP. Local pretreatment with 250 nmol/L of a neurokinin 1 receptor antagonist prevented the SP-induced plasma extravasation, the initiation of CSDs by 10-5 mol/L SP diluted in aqua, and the initiation of CSDs by aqua alone, but did not suppress KCl-induced CSD. Thus neurokinin 1 receptor antagonists may be used to explore the involvement of SP in CSDs in clinical studies.

Combined Use of Unnatural Amino Acids Enables Dual-Color Super-Resolution Imaging of Proteins via Click Chemistry.

Recent advances in optical nanoscopy have brought the imaging resolution to the size of the individual macromolecules, thereby setting stringent requirements for the fluorescent labels. Such requirements are optimally fulfilled by the incorporation of unnatural amino acids (UAAs) in the proteins of interest (POIs), followed by fluorophore conjugation via click chemistry. However, this approach has been limited to single POIs in mammalian cells. Here we solve this problem by incorporating different UAAs in different POIs, which are expressed in independent cell sets. The cells are then fused, thereby combining the different proteins and organelles, and are easily imaged by dual-color super-resolution microscopy. This procedure, which we termed Fuse2Click, is simple, requires only the well-established Amber codon, and allows the use of all previously optimized UAAs and tRNA/RS pairs. This should render it a tool of choice for multicolor click-based imaging.

Synthesis of enantiopure antiobesity drug lorcaserin.

Acylation of enantiomerically pure (R)-2-(3-chlorophenyl)propan-1-amine using chloroacetyl chloride, followed by borane reduction and aluminum chloride catalyzed cyclization yielded enantiopure lorcaserin.

Screw pyrolysis technology for sewage sludge treatment.

Sewage sludge quantities have grown continuously since the introduction of the European Directive (UWWTD 91/271/EEC) relating to the treatment of urban wastewater. In the present, most of the sewage sludge is combusted in single fuels incineration plants or is co-fired in waste incineration or coal power plants. The combustion of sewage sludge is a proven technology. Other treatments, such as fluidized bed gasification, were successfully adopted to produce suitable syngas for power production. Besides, the number of large wastewater treatment plants is relatively small compared to the local rural ones. Moreover, alternative technologies are arising with the main target of nutrients recovery, with a special focus on phosphorus. The aforementioned issues, i.e. the small scale (below 1MW) and the nutrients recovery, suggest that pyrolysis in screw reactors may become an attractive alternative technology for sewage sludge conversion, recovery and recycling. In this work, about 100kg of dried sewage sludge from a plant in Germany were processed at the newly developed STYX Reactor, at KIT. The reactor combines the advantages of screw reactors with the high temperature filtration, in order to produce particle and ash free vapors and condensates, respectively. Experiments were carried out at temperatures between 350°C and 500°C. The yield of the char decreased from 66.7wt.% to 53.0wt.%. The same trend was obtained for the energy yield, while the maximum pyrolysis oil yield of 13.4wt.% was obtained at 500°C. Besides mercury, the metals and the other minerals were completely retained in the char. Nitrogen and sulfur migrated from the solid to the condensate and to the gas, respectively. Based on the energy balance, a new concept for the decentral production of char as well as heat and power in an externally fired micro gas turbine showed a cogeneration efficiency up to about 40%.

Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy.

Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA-based protocols. Glyoxal acted faster than PFA, cross-linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA-based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

Cation selectivity of the presequence translocase channel Tim23 is crucial for efficient protein import.

Virtually all mitochondrial matrix proteins and a considerable number of inner membrane proteins carry a positively charged, N-terminal presequence and are imported by the TIM23 complex (presequence translocase) located in the inner mitochondrial membrane. The voltage-regulated Tim23 channel constitutes the actual protein-import pore wide enough to allow the passage of polypeptides with a secondary structure. In this study, we identify amino acids important for the cation selectivity of Tim23. Structure based mutants show that selectivity is provided by highly conserved, pore-lining amino acids. Mutations of these amino acid residues lead to reduced selectivity properties, reduced protein import capacity and they render the Tim23 channel insensitive to substrates. We thus show that the cation selectivity of the Tim23 channel is a key feature for substrate recognition and efficient protein import.

The continuum of spreading depolarizations in acute cortical lesion development: Examining Leão's legacy.

A modern understanding of how cerebral cortical lesions develop after acute brain injury is based on Aristides Leão's historic discoveries of spreading depression and asphyxial/anoxic depolarization. Treated as separate entities for decades, we now appreciate that these events define a continuum of spreading mass depolarizations, a concept that is central to understanding their pathologic effects. Within minutes of acute severe ischemia, the onset of persistent depolarization triggers the breakdown of ion homeostasis and development of cytotoxic edema. These persistent changes are diagnosed as diffusion restriction in magnetic resonance imaging and define the ischemic core. In delayed lesion growth, transient spreading depolarizations arise spontaneously in the ischemic penumbra and induce further persistent depolarization and excitotoxic damage, progressively expanding the ischemic core. The causal role of these waves in lesion development has been proven by real-time monitoring of electrophysiology, blood flow, and cytotoxic edema. The spreading depolarization continuum further applies to other models of acute cortical lesions, suggesting that it is a universal principle of cortical lesion development. These pathophysiologic concepts establish a working hypothesis for translation to human disease, where complex patterns of depolarizations are observed in acute brain injury and appear to mediate and signal ongoing secondary damage.

Effects of interleukin-1ß on cortical spreading depolarization and cerebral vasculature.

During brain damage and ischemia, the cytokine interleukin-1ß is rapidly upregulated due to activation of inflammasomes. We studied whether interleukin-1ß influences cortical spreading depolarization, and whether lipopolysaccharide, often used for microglial stimulation, influences cortical spreading depolarizations. In anaesthetized rats, cortical spreading depolarizations were elicited by microinjection of KCl. Interleukin-1ß, the IL-1 receptor 1 antagonist, the GABAA receptor blocker bicuculline, and lipopolysaccharide were administered either alone or combined (interleukin-1ß + IL-1 receptor 1 antagonist; interleukin-1ß + bicuculline; lipopolysaccharide + IL-1 receptor 1 antagonist) into a local cortical treatment area. Using microelectrodes, cortical spreading depolarizations were recorded in a non-treatment and in the treatment area. Plasma extravasation in cortical grey matter was assessed with Evans blue. Local application of interleukin-1ß reduced cortical spreading depolarization amplitudes in the treatment area, but not at a high dose. This reduction was prevented by IL-1 receptor 1 antagonist and by bicuculline. However, interleukin-1ß induced pronounced plasma extravasation independently on cortical spreading depolarizations. Application of lipopolysaccharide reduced cortical spreading depolarization amplitudes but prolonged their duration; EEG activity was still present. These effects were also blocked by IL-1 receptor 1 antagonist. Interleukin-1ß evokes changes of neuronal activity and of vascular functions. Thus, although the reduction of cortical spreading depolarization amplitudes at lower doses of interleukin-1ß may reduce deleterious effects of cortical spreading depolarizations, the sum of interleukin-1ß effects on excitability and on the vasculature rather promote brain damaging mechanisms.

TIM29 is a subunit of the human carrier translocase required for protein transport.

Hydrophobic inner mitochondrial membrane proteins with internal targeting signals, such as the metabolite carriers, use the carrier translocase (TIM22 complex) for transport into the inner membrane. Defects in this transport pathway have been associated with neurodegenerative disorders. While the TIM22 complex is well studied in baker's yeast, very little is known about the mammalian TIM22 complex. Using immunoprecipitation, we purified the human carrier translocase and identified a mitochondrial inner membrane protein TIM29 as a novel component, specific to metazoa. We show that TIM29 is a constituent of the 440 kDa TIM22 complex and interacts with oxidized TIM22. Our analyses demonstrate that TIM29 is required for the structural integrity of the TIM22 complex and for import of substrate proteins by the carrier translocase.

N-Palmitoylethanolamine Prevents the Run-down of Amplitudes in Cortical Spreading Depression Possibly Implicating Proinflammatory Cytokine Release.

Cortical spreading depression (CSD), a wave of neuronal depolarization in the cerebral cortex following traumatic brain injury or cerebral ischemia, significantly aggravates brain damage. Here, we tested whether N-palmitoylethanolamine (PEA), a substance that effectively reduces lesion volumes and neurological deficits after ischemic stroke, influences CSD. CSD was elicited chemically in adult rats and occurrence, amplitude, duration and propagation velocity of CSD was determined prior to and for 6 hours after intraperitoneal injection of PEA. The chosen systemic administration of PEA stabilized the amplitude of CSD for at least four hours and prevented the run-down of amplitudes that is typically observed and was also seen in untreated controls. The propagation velocity of the CSD waves was unaltered indicating stable neuronal excitability. The stabilization of CSD amplitudes by PEA indicates that inhibition or prevention of CSD does not underlie PEA's profound neuroprotective effect. Rather, PEA likely inhibits proinflammatory cytokine release thereby preventing the run-down of CSD amplitudes. This contribution of PEA to the maintenance of neuronal excitability in healthy tissue during CSD potentially adds to neuroprotection outside a damaged area, while other mechanisms control PEA-mediated neuroprotection in damaged tissue resulting from traumatic brain injury or cerebral ischemia.

Tumor necrosis factor reduces the amplitude of rat cortical spreading depression in vivo.

OBJECTIVE: Brain damage and ischemia often trigger cortical spreading depression (CSD), which aggravates brain damage. The proinflammatory cytokine tumor necrosis factor (TNF) is significantly upregulated during brain damage, but it is unknown whether TNF influences spreading depression in cerebral cortex in vivo. This question is important because TNF not only furthers inflammatory reactions but might also be neuroprotective. Here we tested the hypothesis that TNF affects CSD, and we explored the direction in which CSD is modified by TNF. METHODS: CSD, elicited by pressure microinjection of KCl, was recorded in anesthetized rats and mice. TNF was administered locally into a trough, providing local TNF treatment of a cortical area. For further analysis, antibodies to TNF receptor (TNFR) 1 or 2 were applied, or CSD was monitored in TNFR1 and TNFR2 knockout mice. γ-Aminobutyric acid (GABA)A receptors were blocked by bicuculline. Immunohistochemistry localized the cortical expression of TNFR1 and TNFR2. RESULTS: Local application of TNF to the cortex reduced dose-dependently the amplitude of CSD. This effect was prevented by blockade or knockout of TNFR2 but not by blockade or knockout of TNFR1. TNFR2 was localized at cortical neurons including parvalbumin-positive inhibitory interneurons, and blockade of GABAA receptors by bicuculline prevented the reduction of CSD amplitudes by TNF. INTERPRETATION: We identified a functional link between TNF and CSD. TNF activates TNFR2 in cortical inhibitory interneurons. The resulting release of GABA reduces CSD amplitudes. In this manner, TNF might be neuroprotective in pathological conditions.