Somatic Depolarization Enhances Hippocampal CA1 Dendritic Spike Propagation and Distal Input-Driven Synaptic Plasticity.

Synaptic inputs that target distal regions of neuronal dendrites can often generate local dendritic spikes that can amplify synaptic depolarization, induce synaptic plasticity, and enhance neuronal output. However, distal dendritic spikes are subject to significant attenuation by dendritic cable properties, and often produce only a weak subthreshold depolarization of the soma. Nonetheless, such spikes have been implicated in memory storage, sensory perception and place field formation. How can such a weak somatic response produce such powerful behavioral effects? Here, we use dual dendritic and somatic recordings in acute hippocampal slices of male mice to reveal that dendritic spike propagation, but not spike initiation, is strongly enhanced when the somatic resting potential is depolarized, likely as a result of increased inactivation of A-type K+ channels. Somatic depolarization also facilitates the induction of a form of dendritic spike driven heterosynaptic plasticity that enhances memory specificity. Thus, the effect of somatic membrane depolarization to enhance dendritic spike propagation and long-term synaptic plasticity is likely to play an important role in hippocampal-dependent spatial representations as well as learning and memory.SIGNIFICANCE STATEMENT Neurons receive synaptic input along their dendrites but produce action potential (AP) output at their soma. Signals arriving at the distal dendrites of pyramidal neurons (PNs) have little impact on the soma unless they combine to initiate a dendritic spike, which needs to propagate to the soma to trigger an AP. This study shows that small subthreshold depolarization of the soma powerfully enhances the propagation of dendritic spikes, through inactivation of dendritic A-type potassium channels. Enhanced dendritic spike propagation also markedly facilitates the induction of a form of plasticity driven by the distal synaptic inputs. Thus, small changes in somatic membrane potential, similar to those observed in vivo, act as a powerful gate of neuronal information transfer.

Enkephalin release from VIP interneurons in the hippocampal CA2/3a region mediates heterosynaptic plasticity and social memory.

The hippocampus contains a diverse array of inhibitory interneurons that gate information flow through local cortico-hippocampal circuits to regulate memory storage. Although most studies of interneurons have focused on their role in fast synaptic inhibition mediated by GABA release, different classes of interneurons express unique sets of neuropeptides, many of which have been shown to exert powerful effects on neuronal function and memory when applied pharmacologically. However, relatively little is known about whether and how release of endogenous neuropeptides from inhibitory cells contributes to their behavioral role in regulating memory formation. Here we report that vasoactive intestinal peptide (VIP)-expressing interneurons participate in social memory storage by enhancing information transfer from hippocampal CA3 pyramidal neurons to CA2 pyramidal neurons. Notably, this action depends on release of the neuropeptide enkephalin from VIP neurons, causing long-term depression of feedforward inhibition onto CA2 pyramidal cells. Moreover, VIP neuron activity in the CA2 region is increased selectively during exploration of a novel conspecific. Our findings, thus, enhance our appreciation of how GABAergic neurons can regulate synaptic plasticity and mnemonic behavior by demonstrating that such actions can be mediated by release of a specific neuropeptide, rather than through classic fast inhibitory transmission.

Routine data-based quality indicators for the treatment of gonarthrosis and coxarthrosis patients in the ambulatory care sector - A study protocol for a cluster-randomised pilot trial to evaluate the MobilE-ARTH study.

BACKGROUND: In 2019, Germany had the highest rate of hip replacement surgery and the fourth highest rate of knee replacement surgery among more than 30 OECD countries. The age-standardised rates were estimated at 174 hip joint and 137 knee joint replacements per 100,000 population. Against this background, the contrast between financial incentives for surgery and missing incentives for non-surgical treatment options is repeatedly discussed. Quality indicators (QIs) can serve to measure and transparently present the quality of evidence-based care. Comparing results in the form of audit and feedback has been shown to improve e.g. guideline-compliant ambulatory care. Existing QIs targeting the care of gon- and coxarthrosis mainly focus on discharge management after joint replacement surgery and/or require additional data collection. Therefore, as part of the MobilE-ARTH project, a set of QIs for ambulatory care prior to joint replacement surgery calculable based on routine data is being developed. The present study's aim is to evaluate the impact of this QI set in terms of providing feedback on the quality of care. METHODS: The MobilE-ARTH project comprises (Phase 1) developing a QI set following the RAND/UCLA Appropriateness Method, (Phase 2) implementing the QIs in established physician networks of a German statutory health insurance (SHI) within a prospective, non-blinded, cluster-randomised pilot study, and (Phase 3) evaluating the QI set's effectiveness. The physicians in the intervention networks will (a) receive feedback reports providing information about the routine data-based QIs of their gon- and/or coxarthrosis patients and aggregated results for their network, and (b) be invited to two voluntary, facilitated network meetings. In these network meetings, the physicians can use the information provided on the feedback reports to discuss multiprofessional care pathways for patients with gon- and/or coxarthrosis. Selected indicators of the QI set will serve as primary and secondary outcome measures. Routine data will be analysed within multi-level models using an intention-to-treat approach. DISCUSSION: Feedback reports help maintaining clinical standards and closing the gap between evidence and medical practice, thus enabling an overall improvement in health care. Providing physicians with QI-based information on quality of care promotes identifying strengths and weaknesses in medical treatments. TRIAL REGISTRATION: German Clinical Trials Register, number DRKS00027516 , Registered 25th January 2022 - Prospectively registered.

Paradoxical Excitatory Impact of SK Channels on Dendritic Excitability.

Dendritic excitability regulates how neurons integrate synaptic inputs and thereby influences neuronal output. As active dendritic events are associated with significant calcium influx they are likely to be modulated by calcium-dependent processes, such as calcium-activated potassium channels. Here we investigate the impact of small conductance calcium-activated potassium channels (SK channels) on dendritic excitability in male and female rat cortical pyramidal neurons in vitro and in vivo Using local applications of the SK channel antagonist apamin in vitro, we show that blocking somatic SK channels enhances action potential output, whereas blocking dendritic SK channels paradoxically reduces the generation of dendritic calcium spikes and associated somatic burst firing. Opposite effects were observed using the SK channel enhancer NS309. The effect of apamin on dendritic SK channels was occluded when R-type calcium channels were blocked, indicating that the inhibitory impact of apamin on dendritic calcium spikes involved R-type calcium channels. Comparable effects were observed in vivo Intracellular application of apamin via the somatic whole-cell recording pipette reduced the medium afterhyperpolarization and increased action potential output during UP states. In contrast, extracellular application of apamin to the cortical surface to block dendritic SK channels shifted the distribution of action potentials within UP states from an initial burst to a more distributed firing pattern, while having no impact on overall action potential firing frequency or UP and DOWN states. These data indicate that somatic and dendritic SK channels have opposite effects on neuronal excitability, with dendritic SK channels counter-intuitively promoting rather than suppressing neuronal output.SIGNIFICANCE STATEMENT Neurons typically receive input from other neurons onto processes called dendrites, and use electrical events such as action potentials for signaling. As electrical events in neurons are usually associated with calcium influx they can be regulated by calcium-dependent processes. One such process is through the activation of calcium-dependent potassium channels, which usually act to reduce action potential signaling. Although this is the case for calcium-dependent potassium channels found at the cell body, we show here that calcium-dependent potassium channels in dendrites of cortical pyramidal neurons counter-intuitively promote rather than suppress action potential output.

The AibR-isovaleryl coenzyme A regulator and its DNA binding site - a model for the regulation of alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus.

Isovaleryl coenzyme A (IV-CoA) is an important building block of iso-fatty acids. In myxobacteria, IV-CoA is essential for the formation of signaling molecules involved in fruiting body formation. Leucine degradation is the common source of IV-CoA, but a second, de novo biosynthetic route to IV-CoA termed AIB (alternative IV-CoA biosynthesis) was recently discovered in M. xanthus. The AIB-operon contains the TetR-like transcriptional regulator AibR, which we characterize in this study. We demonstrate that IV-CoA binds AibR with micromolar affinity and show by gelshift experiments that AibR interacts with the promoter region of the AIB-operon once IV-CoA is present. We identify an 18-bp near-perfect palindromic repeat as containing the AibR operator and provide evidence that AibR also controls an additional genomic locus coding for a putative acetyl-CoA acetyltransferase. To elucidate atomic details, we determined crystal structures of AibR in the apo, the IV-CoA- and the IV-CoA-DNA-bound state to 1.7 Å, 2.35 Å and 2.92 Å, respectively. IV-CoA induces partial unfolding of an α-helix, which allows sequence-specific interactions between AibR and its operator. This study provides insights into AibR-mediated regulation and shows that AibR functions in an unusual TetR-like manner by blocking transcription not in the ligand-free but in the effector-bound state.

AibA/AibB Induces an Intramolecular Decarboxylation in Isovalerate Biosynthesis by Myxococcus xanthus.

Isovaleryl coenzyme A (IV-CoA) is an important precursor for iso-fatty acids and lipids. It acts in the development of myxobacteria, which can produce this compound from acetyl-CoA through alternative IV-CoA biosynthesis (aib). A central reaction of aib is catalyzed by AibA/AibB, which acts as a cofactor-free decarboxylase despite belonging to the family of CoA-transferases. We developed an efficient expression system for AibA/AibB that allowed the determination of high-resolution crystal structures in complex with different ligands. Through mutational studies, we show that an active-site cysteine previously proposed to be involved in decarboxylation is not required for activity. Instead, AibA/AibB seems to induce an intramolecular decarboxylation by binding its substrate in a hydrophobic cavity and forcing it into a bent conformation. Our study opens opportunities for synthetic biology studies, since AibA/AibB may be suitable for the production of isobutene, a precursor of biofuels and chemicals.

Impact of calcium-activated potassium channels on NMDA spikes in cortical layer 5 pyramidal neurons.

Active electrical events play an important role in shaping signal processing in dendrites. As these events are usually associated with an increase in intracellular calcium, they are likely to be under the control of calcium-activated potassium channels. Here, we investigate the impact of calcium-activated potassium channels onN-methyl-d-aspartate (NMDA) receptor-dependent spikes, or NMDA spikes, evoked by glutamate iontophoresis onto basal dendrites of cortical layer 5 pyramidal neurons. We found that small-conductance calcium-activated potassium channels (SK channels) act to reduce NMDA spike amplitude but at the same time, also decrease the iontophoretic current required for their generation. This SK-mediated decrease in NMDA spike threshold was dependent on R-type voltage-gated calcium channels and indicates a counterintuitive, excitatory effect of SK channels on NMDA spike generation, whereas the capacity of SK channels to suppress NMDA spike amplitude is in line with the expected inhibitory action of potassium channels on dendritic excitability. Large-conductance calcium-activated potassium channels had no significant impact on NMDA spikes, indicating that these channels are either absent from basal dendrites or not activated by NMDA spikes. These experiments reveal complex and opposing interactions among NMDA receptors, SK channels, and voltage-gated calcium channels in basal dendrites of cortical layer 5 pyramidal neurons during NMDA spike generation, which are likely to play an important role in regulating the way these neurons integrate the thousands of synaptic inputs they receive.

The Structure of LiuC, a 3-Hydroxy-3-Methylglutaconyl CoA Dehydratase Involved in Isovaleryl-CoA Biosynthesis in Myxococcus xanthus, Reveals Insights into Specificity and Catalysis.

Myxobacteria are able to produce the important metabolite isovaleryl coenzyme A by a route other than leucine degradation. The first step into this pathway is mediated by LiuC, a member of the 3-methylglutaconyl CoA hydratases (MGCH). Here we present crystal structures refined to 2.05 and 1.1 Šof LiuC in the apo form and bound to coenzyme A, respectively. By using simulated annealing we modeled the enzyme substrate complex and identified residues potentially involved in substrate binding, specificity, and catalysis. The dehydration of 3-hydroxy-3-methylglutaconyl CoA to 3-methylglutaconyl CoA catalyzed by LiuC involves Glu112 and Glu132 and likely employs the typical crotonase acid-base mechanism. In this, Tyr231 and Arg69 are key players in positioning the substrate to enable catalysis. Surprisingly, LiuC shows higher sequence and structural similarity to human MGCH than to bacterial forms, although they convert the same substrate. This study provides structural insights into the alternative isovaleryl coenzyme A biosynthesis pathway and might open a path for biofuel research, as isovaleryl-CoA is a source for isobutene, a precursor for renewable fuels and chemicals.

Crystal Structure of the HMG-CoA Synthase MvaS from the Gram-Negative Bacterium Myxococcus xanthus.

A critical step in bacterial isoprenoid production is the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) catalyzed by HMG-CoA synthase (HMGCS). In myxobacteria, this enzyme is also involved in a recently discovered alternative and acetyl-CoA-dependent isovaleryl CoA biosynthesis pathway. Here we present crystal structures of MvaS, the HMGCS from Myxococcus xanthus, in complex with CoA and acetylated active site Cys115, with the second substrate acetoacetyl CoA and with the product of the condensation reaction, 3-hydroxy-3-methylglutaryl CoA. With these structures, we show that MvaS uses the common HMGCS enzymatic mechanism and provide evidence that dimerization plays a role in the formation and stability of the active site. Overall, MvaS shows features typical of the eukaryotic HMGCS and exhibits differences from homologues from Gram-positive bacteria. This study provides insights into myxobacterial alternative isovaleryl CoA biosynthesis and thereby extends the toolbox for the biotechnological production of renewable fuel and chemicals.

Dendritic encoding of sensory stimuli controlled by deep cortical interneurons.

The computational power of single neurons is greatly enhanced by active dendritic conductances that have a large influence on their spike activity. In cortical output neurons such as the large pyramidal cells of layer 5 (L5), activation of apical dendritic calcium channels leads to plateau potentials that increase the gain of the input/output function and switch the cell to burst-firing mode. The apical dendrites are innervated by local excitatory and inhibitory inputs as well as thalamic and corticocortical projections, which makes it a formidable task to predict how these inputs influence active dendritic properties in vivo. Here we investigate activity in populations of L5 pyramidal dendrites of the somatosensory cortex in awake and anaesthetized rats following sensory stimulation using a new fibre-optic method for recording dendritic calcium changes. We show that the strength of sensory stimulation is encoded in the combined dendritic calcium response of a local population of L5 pyramidal cells in a graded manner. The slope of the stimulus-response function was under the control of a particular subset of inhibitory neurons activated by synaptic inputs predominantly in L5. Recordings from single apical tuft dendrites in vitro showed that activity in L5 pyramidal neurons disynaptically coupled via interneurons directly blocks the initiation of dendritic calcium spikes in neighbouring pyramidal neurons. The results constitute a functional description of a cortical microcircuit in awake animals that relies on the active properties of L5 pyramidal dendrites and their very high sensitivity to inhibition. The microcircuit is organized so that local populations of apical dendrites can adaptively encode bottom-up sensory stimuli linearly across their full dynamic range.

Seizures, behavioral deficits, and adverse drug responses in two new genetic mouse models of HCN1 epileptic encephalopathy.

De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo sequence variations in the human HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an impairment in inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the necessity of tailoring effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool toward reaching this objective.

Novel approach to an early assessment of a patient's potential for neurological remission after acute spinal cord injury: Analysis of hemoglobin concentration dynamics.

Context/objective: Examining hemoglobin (Hb) dynamics with regard to the potential of neurological remission in patients with traumatic spinal cord injury (TSCI).Design: Prospective Clinical Observational Study.Setting: BG Trauma Centre Ludwigshafen, Department of Paraplegiology, Rhineland-Palatinate, Germany.Methods: From 2011 to 2017 a total of 80 patients with acute spinal injury were enrolled and divided into three groups: initial neurological impairment either with (G1; n = 33) or without subsequent neurological remission (G0; n = 35) and vertebral fractures without initial neurological impairment as control group (C; n = 12). Blood samples were taken for 3 months at 11 time-points after injury. Analyses were performed using routine diagnostics.Outcome measures: Multiple logistic regression was used to determine the prognostic value of Hb regarding neurological remission respecting clinical covariates.Results: Data showed elevated mean Hb concentrations in G1 from the third day to 1 month compared to G0, Hb levels were significantly higher in G1 after 3 days (P = 0.03, G1 > G0). The final multiple logistic regression model based on this data predicting the presence of neurological remission resulted in an AUC (area under the curve) of 80.5% (CI: 67.8%-93.2%) in the ROC (receiver operating characteristic) analysis.Conclusion: Elevated Hb concentrations are associated with a higher likelihood of neurological remission. Elevated concentrations of Hb in G1 compared to G0 over time might be linked to both a better initial oxygen supply response and a decreased ECM (extracellular matrix) degradation highlighting the role of Hb as a valuable biomarker for neural regeneration after TSCI.

Pursuing More Aggressive Timelines in the Surgical Treatment of Traumatic Spinal Cord Injury (TSCI): A Retrospective Cohort Study with Subgroup Analysis.

BACKGROUND: The optimal timing of surgical therapy for traumatic spinal cord injury (TSCI) remains unclear. The purpose of this study is to evaluate the impact of "ultra-early" (<4 h) versus "early" (4-24 h) time from injury to surgery in terms of the likelihood of neurologic recovery. METHODS: The effect of surgery on neurological recovery was investigated by comparing the assessed initial and final values of the American Spinal Injury Association (ASIA) Impairment Scale (AIS). A post hoc analysis was performed to gain insight into different subgroup regeneration behaviors concerning neurological injury levels. RESULTS: Datasets from 69 cases with traumatic spinal cord injury were analyzed. Overall, 19/46 (41.3%) patients of the "ultra-early" cohort saw neurological recovery compared to 5/23 (21.7%) patients from the "early" cohort (p = 0.112). The subgroup analysis revealed differences based on the neurological level of injury (NLI) of a patient. An optimal cutpoint for patients with a cervical lesion was estimated at 234 min. Regarding the prediction of neurological improvement, sensitivity was 90.9% with a specificity of 68.4%, resulting in an AUC (area under the curve) of 84.2%. In thoracically and lumbar injured cases, the estimate was lower, ranging from 284 (thoracic) to 245 min (lumbar) with an AUC of 51.6% and 54.3%. CONCLUSIONS: Treatment within 24 h after TSCI is associated with neurological recovery. Our hypothesis that intervention within 4 h is related to an improvement in the neurological outcome was not confirmed in our collective. In a clinical context, this suggests that after TSCI there is a time frame to get the right patient to the right hospital according to advanced trauma life support (ATLS) guidelines.

Zinc Concentration Dynamics Indicate Neurological Impairment Odds after Traumatic Spinal Cord Injury.

Traumatic Spinal Cord Injury (TSCI) is debilitating and often results in a loss of motor and sensory function caused by an interwoven set of pathological processes. Oxidative stress and inflammatory processes are amongst the critical factors in the secondary injury phase after TSCI. The essential trace element Zinc (Zn) plays a crucial role during this phase as part of the antioxidant defense system. The study aims to determine dynamic patterns in serum Zn concentration in patients with TSCI and test for a correlation with neurological impairment. A total of 42 patients with TSCI were enrolled in this clinical observational study. Serum samples were collected at five different points in time after injury (at admission, and after 4 h, 9 h, 12 h, 24 h, and 3 d). The analysis of the serum Zn concentrations was conducted by total reflection X-ray fluorescence (TXRF). The patients were divided into two groups-a study group S (n = 33) with neurological impairment, including patients with remission (G1, n = 18) and no remission (G0, n = 15) according to a positive AIS (American Spinal Injury Association (ASIA) Impairment Scale) conversion within 3 months after the trauma; and a control group C (n = 9), consisting of subjects with vertebral fractures without neurological impairment. The patient data and serum concentrations were examined and compared by non-parametric test methods to the neurological outcome. The median Zn concentrations in group S dropped within the first 9 h after injury (964 µg/L at admission versus 570 µg/L at 9 h, p < 0.001). This decline was stronger than in control subjects (median of 751 µg/L versus 729 µg/L, p = 0.023). A binary logistic regression analysis including the difference in serum Zn concentration from admission to 9 h after injury yielded an area under the curve (AUC) of 82.2% (CI: 64.0-100.0%) with respect to persistent neurological impairment. Early Zn concentration dynamics differed in relation to the outcome and may constitute a helpful diagnostic indicator for patients with spinal cord trauma. The fast changes in serum Zn concentrations allow an assessment of neurological impairment risk on the first day after trauma. This finding supports strategies for improving patient care by avoiding strong deficits via adjuvant nutritive measures, e.g., in unresponsive patients after trauma.

The Role of Magnesium in the Secondary Phase After Traumatic Spinal Cord Injury. A Prospective Clinical Observer Study.

In the secondary injury phase after traumatic spinal cord injury (TSCI), oxidative stress and neuroinflammatory responses at the site of injury constitute crucial factors controlling damage extent and may serve as potential therapeutic targets. We determined Magnesium (Mg) serum concentration dynamics in context with the potential of neurological remission in patients with TSCI as Mg is suspected to limit the production of reactive oxygen species and reduce lipid peroxidation. A total of 29 patients with acute TSCI were enrolled, and blood samples were drawn over 3 months at 11 time-points and Mg quantification was performed. Patients were divided into those with (G1, n = 18) or without neurological remission (G0, n = 11). Results show a slight drop in Mg level during the first 4 h after injury, then remained almost unchanged in G1, but increased continuously during the first 7 days after injury in G0. At day 7 Mg concentrations in G1 and G0 were significantly different (p = 0.039, G0 > G1). Significant differences were detected between patients in G1 that presented an AIS (ASIA Impairment Scale) conversion of 1 level versus those with more than 1 level (p = 0.014, G1 AIS imp. = +1 > G1 AI imp. > +1). Low and decreasing levels of Mg within the first 7 days are indicative of a high probability of neurological remission, whereas increasing levels are associated with poor neurological outcome.

Relation of selenium status to neuro-regeneration after traumatic spinal cord injury.

INTRODUCTION: The trace element selenium (Se) is crucial for the biosynthesis of selenoproteins. Both neurodevelopment and the survival of neurons that are subject to stress depend on a regular selenoprotein biosynthesis and sufficient Se supply by selenoprotein P (SELENOP). HYPOTHESIS: Neuro-regeneration after traumatic spinal cord injury (TSCI) is related to the Se status. STUDY DESIGN: Single-centre prospective observational study. PATIENTS AND METHODS: Three groups of patients with comparable injuries were studied; vertebral fractures without neurological impairment (n = 10, group C), patients with TSCI showing no remission (n = 9, group G0), and patients with remission developing positive abbreviated injury score (AIS) conversion within 3 months (n = 10, group G1). Serum samples were available from different time points (upon admission, and after 4, 9 and 12 h, 1 and 3 days, 1 and 2 weeks, and 1, 2 and 3 months). Serum trace element concentrations were determined by total reflection X-ray fluorescence, SELENOP by ELISA, and further parameters by laboratory routine. RESULTS: Serum Se and SELENOP concentrations were higher on admission in the remission group (G1) as compared to G0. During the first week, both parameters remained constant in C and G0, whereas they declined significantly in the remission group. Similarly, the concentration changes between admission and 24 h were most pronounced in this group of recovering patients (G1). Binary logistic regression analysis including the delta of Se and SELENOP within the first 24 h indicated an AUC of 90.0% (CI: 67.4%-100.0%) with regards to predicting the outcome after TSCI. CONCLUSION: A Se deficit might constitute a risk factor for poor outcome after TSCI. A dynamic decline of serum Se and SELENOP concentrations after admission may reflect ongoing repair processes that are associated with higher odds for a positive clinical outcome.

Structural, mechanistic and functional insight into gliotoxin bis-thiomethylation in Aspergillus fumigatus.

Gliotoxin is an epipolythiodioxopiperazine (ETP) class toxin, contains a disulfide bridge that mediates its toxic effects via redox cycling and is produced by the opportunistic fungal pathogen Aspergillus fumigatus Self-resistance against gliotoxin is effected by the gliotoxin oxidase GliT, and attenuation of gliotoxin biosynthesis is catalysed by gliotoxin S-methyltransferase GtmA. Here we describe the X-ray crystal structures of GtmA-apo (1.66 Å), GtmA complexed to S-adenosylhomocysteine (1.33 Å) and GtmA complexed to S-adenosylmethionine (2.28 Å), providing mechanistic insights into this important biotransformation. We further reveal that simultaneous elimination of the ability of A. fumigatus to dissipate highly reactive dithiol gliotoxin, via deletion of GliT and GtmA, results in the most significant hypersensitivity to exogenous gliotoxin observed to date. Indeed, quantitative proteomic analysis of ΔgliT::ΔgtmA reveals an uncontrolled over-activation of the gli-cluster upon gliotoxin exposure. The data presented herein reveal, for the first time, the extreme risk associated with intracellular dithiol gliotoxin biosynthesis-in the absence of an efficient dismutation capacity. Significantly, a previously concealed protective role for GtmA and functionality of ETP bis-thiomethylation as an ancestral protection strategy against dithiol compounds is now evident.

The Impact of BK Channels on Cellular Excitability Depends on their Subcellular Location.

Large conductance calcium-activated potassium channels (or BK channels) fulfil a multitude of roles in the central nervous system. At the soma of many neuronal cell types they control the speed of action potential (AP) repolarization and therefore they can have an impact on neuronal excitability. Due to their presence in nerve terminals they also regulate transmitter release. BK channels have also been shown to be present in the dendrites of some neurons where they can regulate the magnitude and duration of dendritic spikes. Here, we investigate the impact of modulating the activation of BK channels at different locations on the cellular excitability of cortical layer 5 pyramidal neurons. We find that while somatic BK channels help to repolarize APs at the soma and mediate the fast after-hyperpolarization, dendritic BK channels are responsible for repolarization of dendritic calcium spikes and thereby regulate somatic AP burst firing. We found no evidence for a role of dendritic BK channels in the regulation of backpropagating AP amplitude or duration. These experiments highlight the diverse roles of BK channels in regulating neuronal excitability and indicate that their functional impact depends on their subcellular location.

Crystal structure of AibC, a reductase involved in alternative de novo isovaleryl coenzyme A biosynthesis in Myxococcus xanthus.

Isovaleryl coenzyme A (IV-CoA) performs a crucial role during development and fruiting-body formation in myxobacteria, which is reflected in the existence of a de novo biosynthetic pathway that is highly upregulated when leucine, the common precursor of IV-CoA, is limited. The final step in de novo IV-CoA biosynthesis is catalyzed by AibC, a medium-chain dehydrogenase/reductase. Here, the crystal structure of AibC from Myxococcus xanthus refined to 2.55 Šresolution is presented. The protein adopts two different conformations in the crystal lattice, which is a consequence of partial interaction with the purification tag. Based on this structure, it is suggested that AibC most probably uses a Zn(2+)-supported catalytic mechanism in which NADPH is preferred over NADH. Taken together, this study reveals structural details of the alternative IV-CoA-producing pathway in myxobacteria, which may serve as a base for further biotechnological research and biofuel production.