Golexanolone improves fatigue, motor incoordination and gait and memory in rats with bile duct ligation

\ua9 2023 The Authors. Liver International published by John Wiley & Sons Ltd.Background and Aims: Many patients with the chronic cholestatic liver disease primary biliary cholangitis (PBC) show fatigue and cognitive impairment that reduces their quality of life. Likewise, rats with bile duct ligation (BDL) are a model of cholestatic liver disease. Current PBC treatments do not improve symptomatic alterations such as fatigue or cognitive impairment and new, more effective treatments are therefore required. Golexanolone reduces the potentiation of GABAA receptors activation by neurosteroids. Golexanolone reduces peripheral inflammation and neuroinflammation and improves cognitive and motor function in rats with chronic hyperammonemia. The aims of the present study were to assess if golexanolone treatment improves fatigue and cognitive and motor function in cholestatic BDL rats and if this is associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. Methods: Rats were subjected to bile duct ligation. One week after surgery, oral golexanolone was administered daily to BDL and sham-operated controls. Fatigue was analysed in the treadmill, motor coordination in the motorater, locomotor gait in the Catwalk, and short-term memory in the Y-maze. We also analysed peripheral inflammation, neuroinflammation, and GABAergic neurotransmission markers by immunohistochemistry and Western blot. Results: BDL induces fatigue, impairs memory and motor coordination, and alters locomotor gait in cholestatic rats. Golexanolone improves these alterations, and this was associated with improvement of peripheral inflammation, neuroinflammation, and GABAergic neurotransmission in the cerebellum. Conclusion: Golexanolone may have beneficial effects to treat fatigue, and motor and cognitive impairment in patients with the chronic cholestatic liver disease PBC

An incremental approach to automated protein localisation

Tscherepanow M, Jensen N, Kummert F. An incremental approach to automated protein localisation. BMC Bioinformatics. 2008;9(1): 445.Background: The subcellular localisation of proteins in intact living cells is an important means for gaining information about protein functions. Even dynamic processes can be captured, which can barely be predicted based on amino acid sequences. Besides increasing our knowledge about intracellular processes, this information facilitates the development of innovative therapies and new diagnostic methods. In order to perform such a localisation, the proteins under analysis are usually fused with a fluorescent protein. So, they can be observed by means of a fluorescence microscope and analysed. In recent years, several automated methods have been proposed for performing such analyses. Here, two different types of approaches can be distinguished: techniques which enable the recognition of a fixed set of protein locations and methods that identify new ones. To our knowledge, a combination of both approaches – i.e. a technique, which enables supervised learning using a known set of protein locations and is able to identify and incorporate new protein locations afterwards – has not been presented yet. Furthermore, associated problems, e.g. the recognition of cells to be analysed, have usually been neglected. Results: We introduce a novel approach to automated protein localisation in living cells. In contrast to well-known techniques, the protein localisation technique presented in this article aims at combining the two types of approaches described above: After an automatic identification of unknown protein locations, a potential user is enabled to incorporate them into the pre-trained system. An incremental neural network allows the classification of a fixed set of protein location as well as the detection, clustering and incorporation of additional patterns that occur during an experiment. Here, the proposed technique achieves promising results with respect to both tasks. In addition, the protein localisation procedure has been adapted to an existing cell recognition approach. Therefore, it is especially well-suited for high-throughput investigations where user interactions have to be avoided. Conclusion: We have shown that several aspects required for developing an automatic protein localisation technique – namely the recognition of cells, the classification of protein distribution patterns into a set of learnt protein locations, and the detection and learning of new locations – can be combined successfully. So, the proposed method constitutes a crucial step to render image-based protein localisation techniques amenable to large-scale experiments

Structures of insect Imp-L2 suggest an alternative strategy for regulating the bioavailability of insulin-like hormones

The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1–6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers

Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems - Purification of the human type 1 11 beta-hydroxysteroid dehydrogenase

Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound I I p-hydroxysteroid dehydrogenase type I (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved. (C) 2004 Elsevier B.V. All rights reserved

Neuroinflammation alters GABAergic neurotransmission in hyperammonemia and hepatic encephalopathy, leading to motor incoordination. Mechanisms and therapeutic implications

Enhanced GABAergic neurotransmission contributes to impairment of motor coordination and gait and of cognitive function in different pathologies, including hyperammonemia and hepatic encephalopathy. Neuroinflammation is a main contributor to enhancement of GABAergic neurotransmission through increased activation of different pathways. For example, enhanced activation of the TNFα–TNFR1-NF-κB-glutaminase-GAT3 pathway and the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway in cerebellum of hyperammonemic rats enhances GABAergic neurotransmission. This is mediated by mechanisms affecting GABA synthesizing enzymes GAD67 and GAD65, total and extracellular GABA levels, membrane expression of GABAA receptor subunits, of GABA transporters GAT1 and GAT three and of chloride co-transporters. Reducing neuroinflammation reverses these changes, normalizes GABAergic neurotransmission and restores motor coordination. There is an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other and altogether modulate motor coordination and cognitive function. In this way, neuroinflammation may be also reduced by reducing GABAergic neurotransmission, which may also improve cognitive and motor function in pathologies associated to neuroinflammation and enhanced GABAergic neurotransmission such as hyperammonemia, hepatic encephalopathy or Parkinson’s disease. This provides therapeutic targets that may be modulated to improve cognitive and motor function and other alterations such as fatigue in a wide range of pathologies. As a proof of concept it has been shown that antagonists of GABAA receptors such as bicuculline reduces neuroinflammation and improves cognitive and motor function impairment in rat models of hyperammonemia and hepatic encephalopathy. Antagonists of GABAA receptors are not ideal therapeutic tools because they can induce secondary effects. As a more effective treatment to reduce GABAergic neurotransmission new compounds modulating it by other mechanisms are being developed. Golexanolone reduces GABAergic neurotransmission by reducing the potentiation of GABAA receptor activation by neurosteroids such as allopregnanolone. Golexanolone reduces neuroinflammation and GABAergic neurotransmission in animal models of hyperammonemia, hepatic encephalopathy and cholestasis and this is associated with improvement of fatigue, cognitive impairment and motor incoordination. This type of compounds may be useful therapeutic tools to improve cognitive and motor function in different pathologies associated with neuroinflammation and increased GABAergic neurotransmission