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Background: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy., Methods: Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging., Results: We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca 2+ -induced Ca 2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca 2+ channels and ryanodine receptor Ca 2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components., Conclusions: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload., Competing Interests: None.

The structure and biomass of aquatic invertebrate communities play a crucial role in the matter dynamics of streams. However, biomass is rarely quantified in ecological assessments of streams, and little is known about the environmental and anthropogenic factors that influence it. In this study, we aimed to identify environmental factors that are associated with invertebrate structure and biomass through a monitoring of 25 streams across Germany. We identified invertebrates, assigned them to taxonomic and trait-based groups, and quantified biomass using image-based analysis. We found that insecticide pressure generally reduced the abundance of insecticide-vulnerable populations (R 2  = 0.43 applying SPEAR pesticides indicator), but not invertebrate biomass. In contrast, herbicide pressure reduced the biomass of several biomass aggregations. Especially, insecticide-sensitive populations, that were directly (algae feeder, R 2  = 0.39) or indirectly (predators, R 2  = 0.29) dependent on algae, were affected. This indicated a combined effect of possible food shortage due to herbicides and direct insecticide pressure. Specifically, all streams with increased herbicide pressure showed a reduced overall biomass share of Trichoptera from 43 % to 3 % and those of Ephemeroptera from 20 % to 3 % compared to streams grouped by low herbicide pressure. In contrast, insecticide-insensitive Gastropoda increased from 10 % to 45 %, and non-vulnerable leaf-shredding Crustacea increased from 10 % to 22 %. In summary, our results indicate that at the community level, the direct effects of insecticides and the indirect, food-mediated effects of herbicides exert a combined effect on the biomass of sensitive insect groups, thus disrupting food chains at ecosystem level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: SLC4A10 encodes a plasma membrane-bound transporter, which mediates Na + -dependent HCO 3 - import, thus mediating net acid extrusion. Slc4a10 knockout mice show collapsed brain ventricles, an increased seizure threshold, mild behavioral abnormalities, impaired vision, and deafness., Methods: Utilizing exome/genome sequencing in families with undiagnosed neurodevelopmental disorders and international data sharing, 11 patients from 6 independent families with biallelic variants in SLC4A10 were identified. Clinico-radiological and dysmorphology assessments were conducted. A minigene assay, localization studies, intracellular pH recordings, and protein modeling were performed to study the possible functional consequences of the variant alleles., Results: The families harbor 8 segregating ultra-rare biallelic SLC4A10 variants (7 missense and 1 splicing). Phenotypically, patients present with global developmental delay/intellectual disability and central hypotonia, accompanied by variable speech delay, microcephaly, cerebellar ataxia, facial dysmorphism, and infrequently, epilepsy. Neuroimaging features range from some non-specific to distinct neuroradiological findings, including slit ventricles and a peculiar form of bilateral curvilinear nodular heterotopia. In silico analyses showed 6 of 7 missense variants affect evolutionarily conserved residues. Functional analyses supported the pathogenicity of 4 of 7 missense variants., Conclusion: We provide evidence that pathogenic biallelic SLC4A10 variants can lead to neurodevelopmental disorders characterized by variable abnormalities of the central nervous system, including altered brain ventricles, thus resembling several features observed in knockout mice., Competing Interests: Conflict of Interest James R. Lupski owns stock in 23andMe and is a paid consultant for Genome International. Rhonda E. Schnur is an employee of GeneDx, LLC. The other authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

The decomposition of allochthonous organic matter, such as leaves, is a crucial ecosystem process in low-order streams. Microbial communities, including fungi and bacteria, colonize allochthonous organic material, break up large molecules, and increase the nutritional value for macroinvertebrates. Environmental variables are known to affect microbial as well as macroinvertebrate communities and alter their ability to decompose organic matter. Studying the relationship between environmental variables and decomposition has mainly been realized using leaves, with the drawbacks of differing substrate composition and consequently between-study variability. To overcome these drawbacks, artificial substrates have been developed, serving as standardizable surrogates. In the present study, we compared microbial and total decomposition of leaves with the standardized substrates of decotabs and, only for microbial decomposition, of cotton strips, across 70 stream sites in a Germany-wide study. Furthermore, we identified the most influential environmental variables for the decomposition of each substrate from a range of 26 variables, including pesticide toxicity, concentrations of nutrients, and trace elements, using stability selection. The microbial as well as total decomposition of the standardized substrates (i.e., cotton strips and decotabs) were weak or not associated with that of the natural substrate (i.e., leaves, r² < 0.01 to r² = 0.04). The decomposition of the two standardized substrates, however, showed a moderate association (r² = 0.21), which is probably driven by their similar composition, with both being made of cellulose. Different environmental variables were identified as the most influential for each of the substrates and the directions of these relationships contrasted between the substrates. Our results imply that these standardized substrates are unsuitable surrogates when investigating the decomposition of allochthonous organic matter in streams. Environ Toxicol Chem 2023;42:2007-2018. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy) 1 . Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons 2 . Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss 3 , interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance., (© 2023. The Author(s).)

Following agricultural application, pesticides can enter streams through runoff during rain events. However, little information is available on the temporal dynamics of pesticide toxicity during the main application period. We investigated pesticide application and large scale in-stream monitoring data from 101 agricultural catchments obtained from a Germany-wide monitoring from April to July in 2018 and 2019. We analysed temporal patterns of pesticide application, in-stream toxicity and exceedances of regulatory acceptable concentrations (RAC) for over 70 pesticides. On a monthly scale from April to July, toxicity to invertebrates and algae/aquatic plants (algae) obtained with event-driven samples (EDS) was highest in May/June. The peak of toxicity towards invertebrates and algae coincided with the peaks of insecticide and herbicide application. Future monitoring, i.e. related to the Water Framework Directive, could be limited to time periods of highest pesticide applications on a seasonal scale. On a daily scale, toxicity to invertebrates from EDS exceeded those of grab samples collected within one day after rainfall by a factor of 3.7. Within two to three days, toxicity in grab samples declined compared to EDS by a factor of ten for invertebrates, and a factor of 1.6 for algae. Thus, toxicity to invertebrates declined rapidly within 1 day after a rainfall event, whereas toxicity to algae remained elevated for up to 4 days. For six pesticides, RAC exceedances could only be detected in EDS. The exceedances of RACs coincided with the peaks in pesticide application. Based on EDS, we estimated that pesticide exposure would need a 37-fold reduction of all analysed pesticides, to meet the German environmental target to keep RAC exceedances below 1 % of EDS. Overall, our study shows a high temporal variability of exposure on a monthly but also daily scale to individual pesticides that can be linked to their period of application and related rain events., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Vegetated buffer strips (VBS) are an effective measure to retain pesticide inputs during rain events. Numerous studies have examined the retention effects of VBS on pesticides. However, no study has addressed on a large scale with event-related peak concentrations how wide the VBS should be to avoid ecological impacts on aquatic life. Here, we investigated for 115 lowland stream sections in Germany the relevance of environmental and physico-chemical parameters to determine the in-stream pesticide concentration and their ecological risks. Based on peak concentrations related to rain events with precipitation amount resulting in VBS relevant surface runoff for 30 of the 115 investigated stream sections (25 to 70 mm/d), we demonstrated that the average width of VBS was the main parameter (R² = 0.38) reducing the pesticide input ratio, indicating a relevant proportion of surface runoff contributing to the total in-stream pesticide concentrations. Additionally, dry ditches within agricultural fields increased pesticide input (R² = 0.31). Generally, substances classified as slightly mobile were better retained by VBS than mobile substances. Other factors including slope, land use and vegetation cover of VBS had only a minor influence. We assessed the ecological risk of in-stream pesticide concentrations by quantifying exceedances of regulatory- (RAC) and field-validated acceptable concentrations (AC field ). We then translated this ecological risk into protective VBS width by calculating the quotient of in-stream concentration and threshold (RQ). We estimate that a VBS width of 18 m is sufficient to meet the RQ ACfield protection goal for 95% of streams. The presence of dry ditches increased the protective VBS width to 32 m. In current agricultural practice, however, 26% of the water stretches investigated do not comply with the prescribed 5 m VBS. An extension of the VBS area to 18 m would demand 3.8% of agricultural land within the catchments. A 50% reduction in pesticide use, as required by the European green deal, would still result in 39% (RAC) and 68% (AC field ) of event-related samples being exceeded. Consequently, we see the extension of the VBS width as the most efficient mearsure to sustainably reduce pesticide concentrations in small streams., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Patients with inactive thyroid hormone (TH) transporter MCT8 display intellectual disability due to compromised central TH transport and action. As a therapeutic strategy, application of thyromimetic, MCT8-independent compounds Triac (3,5,3'-triiodothyroacetic acid), and Ditpa (3,5-diiodo-thyropropionic acid) was proposed. Here, we directly compared their thyromimetic potential in Mct8/Oatp1c1 double knock-out mice (Dko) modeling human MCT8 deficiency. Dko mice received either Triac (50 ng/g or 400 ng/g) or Ditpa (400 ng/g or 4000 ng/g) daily during the first three postnatal weeks. Saline-injected Wt and Dko mice served as controls. A second cohort of Dko mice received Triac (400 ng/g) daily between postnatal weeks 3 and 6. Thyromimetic effects were assessed at different postnatal stages by immunofluorescence, ISH, qPCR, electrophysiological recordings, and behavior tests. Triac treatment (400 ng/g) induced normalized myelination, cortical GABAergic interneuron differentiation, electrophysiological parameters, and locomotor performance only when administered during the first three postnatal weeks. Ditpa (4000 ng/g) application to Dko mice during the first three postnatal weeks resulted in normal myelination and cerebellar development but only mildly improved neuronal parameters and locomotor function. Together, Triac is highly-effective and more efficient than Ditpa in promoting CNS maturation and function in Dko mice yet needs to be initiated directly after birth for the most beneficial effects.

Biological indices aim to reflect the ecological quality of streams based on the community's species or trait composition. Accordingly, the capability to predict the ecological quality depends on (i) the knowledge on the association of taxa or traits with stressors and (ii) the taxonomic and quantitative resolution of taxa. Generally speaking, a higher resolution is associated with a better linkage between environmental condition and biological response but also with higher efforts and costs. So far it is unknown how the taxonomic and quantitative resolution affect the ecological quality assessment of streams related to pesticide effects when applying the invertebrate-based indicator SPEAR pesticides . We investigated the ecological quality of 101 streams considering four taxonomic levels (species, genus, family, order) and three quantitative resolutions (abundance, three abundance classes, and presence-absence). In a multiple linear regression analysis between 13 investigated stressors and SPEAR pesticides , the full models' explained variance remained fairly constant with decreasing taxonomic and quantitative resolution. As expected, the highest association between pesticide pressure and SPEAR pesticides was reached at a species/abundance resolution yielding an R 2 of 0.43. In contrast, the lowest quantitative resolution of order level combined with presence-absence information revealed an explained variance of 0.28 R 2 . We suggest the family/abundance class resolution (R 2 = 0.38) as the best trade-off between effort and accuracy for large-scale monitoring. Due to a comparable linear regression at family/abundance class resolution, the assigned ecological quality classes were largely congruent (69 %) to species/abundance resolution. We conclude that the ecological quality assessment with SPEAR pesticides at family/abundance class resolution can be used to link pesticide contamination and invertebrate community structure with less taxonomic expertise and less quantification effort., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

GABA A receptors are ligand-gated ion channels, which are predominantly permeable for chloride. The neuronal K-Cl cotransporter KCC2 lowers the intraneuronal chloride concentration and thus plays an important role for GABA signaling. KCC2 loss-of-function is associated with seizures and epilepsy. Here, we show that KCC2 is expressed in the majority of parvalbumin-positive interneurons (PV-INs) of the mouse brain. PV-INs receive excitatory input from principle cells and in turn control principle cell activity by perisomatic inhibition and inhibitory input from other interneurons. Upon Cre-mediated disruption of KCC2 in mice, the polarity of the GABA response of PV-INs changed from hyperpolarization to depolarization for the majority of PV-INs. Reduced excitatory postsynaptic potential-spike (E-S) coupling and increased spontaneous inhibitory postsynaptic current (sIPSC) frequencies further suggest that PV-INs are disinhibited upon disruption of KCC2. In vivo , PV-IN-specific KCC2 knockout mice display a reduced seizure threshold and develop spontaneous sometimes fatal seizures. We further found a time dependent loss of PV-INs, which was preceded by an up-regulation of pro-apoptotic genes upon disruption of KCC2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Herrmann, Gerth, Dittmann, Pensold, Ungelenk, Liebmann and Hübner.)

The Water Framework Directive (WFD) demands that good status is to be achieved for all European water bodies. While governmental monitoring under the WFD mostly concludes a good status with regard to pesticide pollution, numerous scientific studies have demonstrated widespread negative ecological impacts of pesticide exposure in surface waters. To identify reasons for this discrepancy, we analysed pesticide concentrations measured in a monitoring campaign of 91 agricultural streams in 2018 and 2019 using methodologies that exceed the requirements of the WFD. This included a sampling strategy that takes into account the periodic occurrence of pesticides and a different analyte spectrum designed to reflect current pesticide use. We found that regulatory acceptable concentrations (RACs) were exceeded for 39 different pesticides at 81% of monitoring sites. In comparison, WFD-compliant monitoring of the same sites would have detected only eleven pesticides as exceeding the WFD-based environmental quality standards (EQS) at 35% of monitoring sites. We suggest three reasons for this underestimation of pesticide risk under the WFD-compliant monitoring: (1) The sampling approach - the timing and site selection are unable to adequately capture the periodic occurrence of pesticides and investigate surface waters particularly susceptible to pesticide risks; (2) the measuring method - a too narrow analyte spectrum (6% of pesticides currently approved in Germany) and insufficient analytical capacities result in risk drivers being overlooked; (3) the assessment method for measured concentrations - the protectivity and availability of regulatory thresholds are not sufficient to ensure a good ecological status. We therefore propose practical and legal refinements to improve the WFD's monitoring and assessment strategy in order to gain a more realistic picture of pesticide surface water pollution. This will enable more rapid identification of risk drivers and suitable risk management measures to ultimately improve the status of European surface waters., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pesticide applications in agricultural crops often comprise a mixture of plant protection products (PPP), and single fields face multiple applications per year leading to complex pesticide mixtures in the environment. Restricted to single PPP, the current European Union PPP regulation, however, disregards the ecological risks of pesticide mixtures. To quantify this additional risk, we evaluated the contribution of single pesticide active ingredients to the additive mixture risk for aquatic risk indicators (invertebrates and algae) in 464 different PPP used, 3446 applications sprayed and 830 water samples collected in Central Europe, Germany. We identified an average number of 1.3 different pesticides in a single PPP, 3.1 for complete applications often involving multiple PPP and 30 in stream water samples. Under realistic worst-case conditions, the estimated stream water pesticide risk based on additive effects was 3.2 times higher than predicted from single PPP. We found that in streams, however, the majority of regulatory threshold exceedances was caused by single pesticides alone (69% for algae, 81% for invertebrates). Both in PPP applications and in stream samples, pesticide exposure occurred in repeated pulses each driven by one to few alternating pesticides. The time intervals between pulses were shorter than the 8 weeks considered for ecological recovery in environmental risk assessment in 88% of spray series and 53% of streams. We conclude that pesticide risk assessment should consider an additional assessment factor to account for the additive, but also potential synergistic simultaneous pesticide mixture risk. Additionally, future research and risk assessment need to address the risk from the frequent sequential pesticide exposure observed in this study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Few studies have examined the exposure of small streams (< 30 km 2 catchment size) to agriculturally used pesticides, compared to large rivers. A total of 105 sites in 103 small agricultural streams were investigated for 76 pesticides (insecticides, herbicides, fungicides) and 32 pesticide metabolites in spring and summer over two years (2018 and 2019) during dry weather and rainfall using event-driven sampling. The median total concentration of the 76 pesticides was 0.18 µg/L, with 9 pesticides per sample on average (n = 815). This is significantly higher than monitoring data for larger streams, reflecting the close proximity to agricultural fields and the limited dilution by non-agricultural waters. The frequency of detection of all pesticides correlated with sales quantity and half-lives in water. Terbuthylazine, MCPA, boscalid, and tebuconazole showed the highest median concentrations. The median of the total concentration of the 32 metabolites exceeded the pesticide concentration by more than an order of magnitude. During dry weather, the median total concentration of the 76 pesticides was 0.07 µg/L, with 5 pesticides per sample on average. Rainfall events increased the median total pesticide concentration by a factor of 10 (to 0.7 µg/L), and the average number of pesticides per sample to 14 (with up to 41 in single samples). The concentration increase was particularly strong for 2,4-D, MCPA, terbuthylazine, and nicosulfuron (75 percentile). Metabolite concentrations were generally less responsive to rainfall, except for those of terbuthylazine, flufenacet, metamitron, and prothioconazole. The frequent and widespread exceedance of the regulatory acceptable concentrations (RAC) of the 76 pesticides during both, dry weather and rainfall, suggests that current plant protection product authorization and risk mitigation methods are not sufficient to protect small streams., (Copyright © 2021. Published by Elsevier Ltd.)

Despite elaborate regulation of agricultural pesticides, their occurrence in non-target areas has been linked to adverse ecological effects on insects in several field investigations. Their quantitative role in contributing to the biodiversity crisis is, however, still not known. In a large-scale study across 101 sites of small lowland streams in Central Europe, Germany we revealed that 83% of agricultural streams did not meet the pesticide-related ecological targets. For the first time we identified that agricultural nonpoint-source pesticide pollution was the major driver in reducing vulnerable insect populations in aquatic invertebrate communities, exceeding the relevance of other anthropogenic stressors such as poor hydro-morphological structure and nutrients. We identified that the current authorisation of pesticides, which aims to prevent unacceptable adverse effects, underestimates the actual ecological risk as (i) measured pesticide concentrations exceeded current regulatory acceptable concentrations in 81% of the agricultural streams investigated, (ii) for several pesticides the inertia of the authorisation process impedes the incorporation of new scientific knowledge and (iii) existing thresholds of invertebrate toxicity drivers are not protective by a factor of 5.3 to 40. To provide adequate environmental quality objectives, the authorisation process needs to include monitoring-derived information on pesticide effects at the ecosystem level. Here, we derive such thresholds that ensure a protection of the invertebrate stream community., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.

Mutations in the genes encoding for voltage-gated sodium channels cause profound sensory disturbances and other symptoms dependent on the distribution of a particular channel subtype in different organs. Humans with the gain-of-function mutation p.Leu811Pro in SCN11A (encoding for the voltage-gated Nav1.9 channel) exhibit congenital insensitivity to pain, pruritus, self-inflicted injuries, slow healing wounds, muscle weakness, Charcot-like arthropathies, and intestinal dysmotility. As already shown, knock-in mice (Scn11a+/L799P) carrying the orthologous mutation p.Leu799Pro replicate reduced pain sensitivity and show frequent tissue lesions. In the present study we explored whether Scn11a+/L799P mice develop also pruritus, muscle weakness, and changes in gastrointestinal transit time. Furthermore, we analyzed morphological and functional differences in nerves, skeletal muscle, joints and small intestine from Scn11a+/L799P and Scn11a+/+ wild type mice. Compared to Scn11a+/+ mice, Scn11a+/L799P mice showed enhanced scratching bouts before skin lesions developed, indicating pruritus. Scn11a+/L799P mice exhibited reduced grip strength, but no disturbances in motor coordination. Skeletal muscle fiber types and joint architecture were unaltered in Scn11a+/L799P mice. Their gastrointestinal transit time was unaltered. The small intestine from Scn11a+/L799P showed a small shift towards less frequent peristaltic movements. Similar proportions of lumbar dorsal root ganglion neurons from Scn11a+/L799P and Scn11a+/+ mice were calcitonin gene-related peptide (CGRP-) positive, but isolated sciatic nerves from Scn11a+/L799P mice exhibited a significant reduction of the capsaicin-evoked release of CGRP indicating reduced neurogenic inflammation. These data indicate important Nav1.9 channel functions in several organs in both humans and mice. They support the pathophysiological relevance of increased basal activity of Nav1.9 channels for sensory abnormalities (pain and itch) and suggest resulting malfunctions of the motor system and of the gastrointestinal tract. Scn11a+/L799P mice are suitable to investigate the role of Nav1.9, and to explore the pathophysiological changes and mechanisms which develop as a consequence of Nav1.9 hyperactivity., Competing Interests: Matthias Ebbinghaus was funded by Charles River Discovery Research Services Germany GmbH after the experimental part of this study was finished. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Rain events may impact the chemical pollution burden in rivers. Forty-four small streams in Germany were profiled during several rain events for the presence of 395 chemicals and five types of mixture effects in in vitro bioassays (cytotoxicity; activation of the estrogen, aryl hydrocarbon, and peroxisome proliferator-activated receptors; and oxidative stress response). While these streams were selected to cover a wide range of agricultural impacts, in addition to the expected pesticides, wastewater-derived chemicals and chemicals typical for street runoff were detected. The unexpectedly high estrogenic effects in many samples indicated the impact by wastewater or overflow of combined sewer systems. The 128 water samples exhibited a high diversity of chemical and effect patterns, even for different rain events at the same site. The detected 290 chemicals explained only a small fraction (<8%) of the measured effects. The experimental effects of the designed mixtures of detected chemicals that were expected to dominate the mixture effects of detected chemicals were consistent with predictions for concentration addition within a factor of two for 94% of the mixtures. Overall, the burden of chemicals and effects was much higher than that previously detected in surface water during dry weather, with the effects often exceeding proposed effect-based trigger values.

The balance of excitation and inhibition is essential for cortical information processing, relying on the tight orchestration of the underlying subcellular processes. Dynamic transcriptional control by DNA methylation, catalyzed by DNA methyltransferases (DNMTs), and DNA demethylation, achieved by ten-eleven translocation (TET)-dependent mechanisms, is proposed to regulate synaptic function in the adult brain with implications for learning and memory. However, focus so far is laid on excitatory neurons. Given the crucial role of inhibitory cortical interneurons in cortical information processing and in disease, deciphering the cellular and molecular mechanisms of GABAergic transmission is fundamental. The emerging relevance of DNMT and TET-mediated functions for synaptic regulation irrevocably raises the question for the targeted subcellular processes and mechanisms. In this study, we analyzed the role dynamic DNA methylation has in regulating cortical interneuron function. We found that DNMT1 and TET1/TET3 contrarily modulate clathrin-mediated endocytosis. Moreover, we provide evidence that DNMT1 influences synaptic vesicle replenishment and GABAergic transmission, presumably through the DNA methylation-dependent transcriptional control over endocytosis-related genes. The relevance of our findings is supported by human brain sample analysis, pointing to a potential implication of DNA methylation-dependent endocytosis regulation in the pathophysiology of temporal lobe epilepsy, a disease characterized by disturbed synaptic transmission., (© The Author(s) 2020. Published by Oxford University Press.)

Brain functions are extremely sensitive to pH changes because of the pH-dependence of proteins involved in neuronal excitability and synaptic transmission. Here, we show that the Na+/H+ exchanger Nhe1, which uses the Na+ gradient to extrude H+, is expressed at both inhibitory and excitatory presynapses. We disrupted Nhe1 specifically in mice either in Emx1-positive glutamatergic neurons or in parvalbumin-positive cells, mainly GABAergic interneurons. While Nhe1 disruption in excitatory neurons had no effect on overall network excitability, mice with disruption of Nhe1 in parvalbumin-positive neurons displayed epileptic activity. From our electrophysiological analyses in the CA1 of the hippocampus, we conclude that the disruption in parvalbumin-positive neurons impairs the release of GABA-loaded vesicles, but increases the size of GABA quanta. The latter is most likely an indirect pH-dependent effect, as Nhe1 was not expressed in purified synaptic vesicles itself. Conclusively, our data provide first evidence that Nhe1 affects network excitability via modulation of inhibitory interneurons., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

There is convincing evidence that the deprivation of one sense can lead to adaptive neuronal changes in spared primary sensory cortices. However, the repercussions of late-onset sensory deprivations on functionality of the remaining sensory cortices are poorly understood. Using repeated intrinsic signal imaging we investigated the effects of whisker or auditory deprivation (WD or AD, respectively) on responsiveness of the binocular primary visual cortex (V1) in fully adult mice. The binocular zone of mice is innervated by both eyes, with the contralateral eye always dominating V1 input over ipsilateral eye input, the normal ocular dominance (OD) ratio. Strikingly, we found that 3 days of WD or AD induced a transient shift of OD, which was mediated by a potentiation of V1 input through the ipsilateral eye. This cross-modal effect was accompanied by strengthening of layer 4 synapses in V1, required visual experience through the ipsilateral eye and was mediated by an increase of the excitation/inhibition ratio in V1. Finally, we demonstrate that both WD and AD induced a long-lasting improvement of visual performance. Our data provide evidence that the deprivation of a non-visual sensory modality cross-modally induces experience dependent V1 plasticity and improves visual behavior, even in adult mice., Competing Interests: The authors have declared that no competing interests exist.

It has been demonstrated that sensory deprivation results in homeostatic adjustments recovering neuronal activity of the deprived cortex. For example, deprived vision multiplicatively scales up mEPSC amplitudes in the primary visual cortex, commonly referred to as synaptic scaling. However, whether synaptic scaling also occurs in auditory cortex after auditory deprivation remains elusive. Using periodic intrinsic optical imaging in adult mice, we show that conductive hearing loss (CHL), initially led to a reduction of primary auditory cortex (A1) responsiveness to sounds. However, this was followed by a complete recovery of A1 activity evoked sounds above the threshold for bone conduction, 3 days after CHL. Over the same time course patch-clamp experiments in slices revealed that mEPSC amplitudes in A1 layers 2/3 pyramids scaled up multiplicatively in CHL mice. No recovery of sensory evoked A1 activation was evident in TNFα KO animals, which lack synaptic scaling. Additionally, we could show that the suppressive effect of sounds on visually evoked visual cortex activity completely recovered along with TNFα dependent A1 homeostasis in WT animals. This is the first demonstration of homeostatic multiplicative synaptic scaling in the adult A1. These findings suggest that mild hearing loss massively affects auditory processing in adult A1.

During early development, a substantial proportion of central neurons undergoes programmed cell death. This activity-dependent process is essential for the proper structural and functional development of the brain. To uncover cell type-specific differences in the regulation of neuronal survival versus apoptosis, we studied activity-regulated cell death in Cajal-Retzius neurons (CRNs) and the overall neuronal population in the developing mouse cerebral cortex. CRNs in the upper neocortical layer represent an early-born neuronal population, which is important for cortical development and largely disappears by apoptosis during neonatal stages. In contrast to the overall neuronal population, activity blockade with tetrodotoxin improved survival of CRNs in culture. Activation of GABAA receptors also blocked spontaneous activity and caused overall cell death including apoptosis of CRNs. Blockade of the Na-K-Cl transporter NKCC1 in vitro or its genetic deletion in vivo rescued CRNs from apoptosis. This effect was mediated by blockade of the p75NTR receptor signaling pathway. In summary, we discovered a novel developmental death pathway mediated by NKCC1, via GABAA receptor-mediated membrane depolarization and p75NTR signaling in CRNs. This pathway controls apoptosis of CRNs and may be critically involved in neurodevelopmental disorders such as autism and schizophrenia., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Rer1 is a sorting receptor in the early secretory pathway that controls the assembly and the cell surface transport of selected multimeric membrane protein complexes. Mice with a Purkinje cell (PC) specific deletion of Rer1 showed normal polarization and differentiation of PCs and normal development of the cerebellum. However, PC-specific loss of Rer1 led to age-dependent motor deficits in beam walk, ladder climbing and gait. Analysis of brain sections revealed a specific degeneration of PCs in the anterior cerebellar lobe in old animals. Electrophysiological recordings demonstrated severe deficits in spontaneous action potential generation. Measurements of resurgent currents indicated decreased surface densities of voltage-gated sodium channels (Na v ), but not changes in individual channels. Analysis of mice with a whole brain Rer1-deletion demonstrated a strong down-regulation of Na v 1.6 and 1.1 in the absence of Rer1, whereas protein levels of the related Ca v 2.1 and of K v 3.3 and 7.2 channels were not affected. The data suggest that Rer1 controls the assembly and transport of Na v 1.1 and 1.6, the principal sodium channels responsible for recurrent firing, in PCs.

Stroke survivors often recover from motor deficits, either spontaneously or with the support of rehabilitative training. Since tonic GABAergic inhibition controls network excitability, it may be involved in recovery. Middle cerebral artery occlusion in rodents reduces tonic GABAergic inhibition in the structurally intact motor cortex (M1). Transcript and protein abundance of the extrasynaptic GABAA-receptor complex α4β3δ are concurrently reduced (δ-GABAARs). In vivo and in vitro analyses show that stroke-induced glutamate release activates NMDA receptors, thereby reducing KCC2 transporters and down-regulates δ-GABAARs. Functionally, this is associated with improved motor performance on the RotaRod, a test in which mice are forced to move in a similar manner to rehabilitative training sessions. As an adverse side effect, decreased tonic inhibition facilitates post-stroke epileptic seizures. Our data imply that early and sometimes surprisingly fast recovery following stroke is supported by homeostatic, endogenous plasticity of extrasynaptic GABAA receptors.

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

Slc4a10 is a Na(+)-coupled Cl(-)-HCO3 (-) exchanger, which is expressed in principal and inhibitory neurons as well as in choroid plexus epithelial cells of the brain. Slc4a10 knockout (KO) mice have collapsed brain ventricles and display an increased seizure threshold, while heterozygous deletions in man have been associated with idiopathic epilepsy and other neurological symptoms. To further characterize the role of Slc4a10 for network excitability, we compared input-output relations as well as short and long term changes of evoked field potentials in Slc4a10 KO and wildtype (WT) mice. While responses of CA1 pyramidal neurons to stimulation of Schaffer collaterals were increased in Slc4a10 KO mice, evoked field potentials did not differ between genotypes in the stratum radiatum or the neocortical areas analyzed. Paired pulse facilitation was diminished in the hippocampus upon disruption of Slc4a10. In the neocortex paired pulse depression was increased. Though short term plasticity is modulated via Slc4a10, long term potentiation appears independent of Slc4a10. Our data support that Slc4a10 dampens neuronal excitability and thus sheds light on the pathophysiology of SLC4A10 associated pathologies.

Insights into mechanisms coordinating membrane remodeling, local actin nucleation, and postsynaptic scaffolding during postsynapse formation are important for understanding vertebrate brain function. Gene knockout and RNAi in individual neurons reveal that the F-BAR protein syndapin I is a crucial postsynaptic coordinator in formation of excitatory synapses. Syndapin I deficiency caused significant reductions of synapse and dendritic spine densities. These syndapin I functions reflected direct, SH3 domain-mediated associations and functional interactions with ProSAP1/Shank2. They furthermore required F-BAR domain-mediated membrane binding. Ultra-high-resolution imaging of specifically membrane-associated, endogenous syndapin I at membranes of freeze-fractured neurons revealed that membrane-bound syndapin I preferentially occurred in spines and formed clusters at distinct postsynaptic membrane subareas. Postsynaptic syndapin I deficiency led to reduced frequencies of miniature excitatory postsynaptic currents, i.e., to defects in synaptic transmission phenocopying ProSAP1/Shank2 knockout, and impairments in proper synaptic ProSAP1/Shank2 distribution. Syndapin I-enriched membrane nanodomains thus seem to be important spatial cues and organizing platforms, shaping dendritic membrane areas into synaptic compartments.

The sensation of pain protects the body from serious injury. Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Nav1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system. Mutant Nav1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.

In vitro modelling of neuronal pathologies is, in particular, demanding and a lot of efforts have been undertaken to differentiate skin derived precursor cells into neuronal cells. However, so far all attempts did not result in cells with functional features of neurons like the ability to generate action potentials or synaptic activity. Here, we report that simple modifications of the protocols result in neuronal cells that display action potentials and synaptic activity. We think that our observation is an important step to model individual neuronal pathologies in vitro.

Background: Patients with malignancies often suffer from thrombembolic events that complicate the course of cancer disease and reduce the patients' quality of life or shorten the survival time in severe cases. This phenomenon is also known for patients with primary or secondary brain tumors; but the reasons are not identified., Methods: We performed a prospective case-controlled study of patients with brain metastases but without any active peripheral tumor site. Blood of patients was collected perioperatively and investigated for coagulation factor activities. Moreover, we analyzed the expression of coagulation factors and their receptors within the tumor material of brain metastases from clear-cell renal cell carcinomas and small-cell carcinomas of the lung., Results: Here, we show that even patients without an active peripheral tumor disease that means without any tumor masses outside the central nervous system after anticancer treatment by surgery, radiation therapy, or chemotherapy but with symptomatic brain metastasis develop an increased systemic activation of multiple coagulation factors. The pro-coagulatory state is expressed preoperatively, but also can be observed in the early postoperative period. Additionally to that, intracerebral metastases of clear-cell renal cell carcinomas and of small-cell carcinomas of the lung express prothrombin, thrombin, factor X, and the protease-activated receptors type 1, 2, 3, and 4., Conclusions: These observations support the hypothesis of a link between the hemostatic system in the periphery and the malignant tumor disease even when the tumor is an intracerebral metastasis and the affected patient currently is free of a systemically active tumor. The results of this study support the hypothesis that the concerted action of coagulation factors and their receptors within the metastasis tissue itself and the systemic coagulation system could control the malignant behavior of tumor disease and make larger prospective trials mandatory.

Behavioral and field potential studies suggest that--shortly after stress--noradrenaline and corticosterone interact to affect the function of basolateral amygdala (BLA) neurons. Here, we tested, at the single-cell level, to what extent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated and N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses of identified BLA neurons are affected by relatively low concentrations of the beta-agonist isoproterenol, how this is influenced by concomitant application of corticosterone, and how isoproterenol effects are influenced by corticosterone given several hours in advance. We observed that isoproterenol concentration-dependently enhances AMPA receptor-mediated (but not NMDA receptor-mediated) responses; near-maximal effects were induced by 1 microm isoproterenol. Corticosterone alone did not rapidly affect AMPA and NMDA-mediated responses. NMDA-mediated responses were also not affected by the hormone in a delayed manner; AMPA-mediated responses were slowly suppressed by corticosterone, but only with high stimulation intensities. If corticosterone was co-applied with isoproterenol (0.4 or 1 microm), facilitation of AMPA-mediated responses was comparable to that seen with isoproterenol alone. However, if corticosterone was applied several hours in advance of the beta-agonist, the effect of 0.4 microm isoproterenol on AMPA-mediated responses was reduced. This supports the notion that, in the BLA, isoproterenol facilitates synaptic transmission, a process that can be suppressed by corticosterone in a slow manner. Overall, the data suggest that, despite the previously reported ability of corticosterone to cause long-term increases in excitability in the BLA, the hormone still retains some capacity to slowly exert a normalizing action on local activity.

The stress hormone corticosterone increases the amplitude of the slow afterhyperpolarization (sAHP) in CA1 pyramidal neurons, without affecting resting membrane potential, input resistance, or action potential characteristics. We here examined how corticosterone affects these properties in the basolateral amygdala (BLA). In the amygdala, corticosterone does not change the AHP amplitude, nor any of the passive and active membrane properties studied. The lack of effect on the AHP is surprising since in both areas corticosterone increases high-voltage-activated sustained calcium currents, which supposedly regulate the sAHP. We wondered whether corticosterone targets different calcium channel subunits in the two areas because currents through only one of the subunits (Cav1.3) are thought to alter the AHP amplitude. In situ hybridization studies revealed that CA1 cells express Cav1.2 and Cav1.3 subunits; corticosterone does not transcriptionally regulate Cav1.2 but increases Cav1.3 expression compared with vehicle treatment. In the BLA, Cav1.3 expression was not detectable, both after control and corticosterone treatment. Cav1.2 is moderately expressed. In a modeling study, we examined putative consequences of changes in specific calcium channel subunit expression and calcium extrusion by corticosterone for the AHP in CA1 and amygdala neurons. A differential distribution and transcriptional regulation of Cav1.2 and Cav1.3 in the CA1 area versus BLA partly explain the observed differences in AHP amplitude. The functional implication of these findings could be that stress-induced arousal of activity in the BLA is more prolonged than that in the CA1 hippocampal area, so that information with an emotional component is more effectively encoded.

Large-conductance Ca(2+)-activated K(+) channels (BK channels) are highly expressed in human glioma cells. However, less is known about their biological function in these cells. We used the patch-clamp technique to investigate activation properties of BK channels and time-lapse microscopy to evaluate the role of BK channel activation in migration of 1321N1 human glioma cells. In whole cells, internal perfusion with a solution containing 500 nM free Ca(2+) and external application of the BK channel opener phloretin (100 micro M) shifted the activation threshold of BK channel currents toward more negative voltages of about -30 mV, which is close to the resting potential of the cells. The concentration of intracellular Ca(2+) in fura-2-loaded 1321N1 cells was measured to be 235+/-19 nM and was increased to 472+/-25 nM after treatment with phloretin. Phloretin and another BK channel opener NS1619 (100 micro M) reduced the migration velocity by about 50%. A similar reduction was observed following muscarinic stimulation of glioma cells with acetylcholine (100 micro M). The effects of phloretin, NS1619 and acetylcholine on cell migration were completely abolished by co-application of the specific BK channel blockers paxilline (5 micro M) and iberiotoxin (100 nM). The phloretin-induced increase in intracellular Ca(2+) was unaffected by the removal of extracellular Ca(2+) and co-application of paxilline. These findings indicate that glioma cell migration was inhibited through BK channel activation, independent of intracellular Ca(2+).

The functional role of BK channels, which are consistently expressed in glioma cells, is not clear. Here we show that the BK channels are regularly active in human 1321N1 astrocytoma cells at physiological membrane potentials. The proliferation of the cells at the physiological external [K+] of 5 mM is compared with that at the elevated external [K+] of 20 mM, simulating the situation in rapidly growing, necrotic tumours in vivo. High extracellular [K+] in the range 10-30 mM significantly increases the proliferation of 1321N1 cells. This K+ induced proliferation can be completely abolished by applying the specific BK channel blockers iberiotoxin (IBTX) or 1 mM tetraethylammonium (TEA). Neither blocker has any effect on cell growth at 5 mM [K+]e. These findings indicate a particular role of BK channels in astrocytoma cell proliferation.