Impaired Glymphatic Function and Pulsation Alterations in a Mouse Model of Vascular Cognitive Impairment

Copyright \ua9 2022 Li, Kitamura, Beverley, Koudelka, Duncombe, Lennen, Jansen, Marshall, Platt, Wiegand, Carare, Kalaria, Iliff and Horsburgh. Large vessel disease and carotid stenosis are key mechanisms contributing to vascular cognitive impairment (VCI) and dementia. Our previous work, and that of others, using rodent models, demonstrated that bilateral common carotid stenosis (BCAS) leads to cognitive impairment via gradual deterioration of the neuro-glial-vascular unit and accumulation of amyloid-β (Aβ) protein. Since brain-wide drainage pathways (glymphatic) for waste clearance, including Aβ removal, have been implicated in the pathophysiology of VCI via glial mechanisms, we hypothesized that glymphatic function would be impaired in a BCAS model and exacerbated in the presence of Aβ. Male wild-type and Tg-SwDI (model of microvascular amyloid) mice were subjected to BCAS or sham surgery which led to a reduction in cerebral perfusion and impaired spatial learning acquisition and cognitive flexibility. After 3 months survival, glymphatic function was evaluated by cerebrospinal fluid (CSF) fluorescent tracer influx. We demonstrated that BCAS caused a marked regional reduction of CSF tracer influx in the dorsolateral cortex and CA1-DG molecular layer. In parallel to these changes increased reactive astrogliosis was observed post-BCAS. To further investigate the mechanisms that may lead to these changes, we measured the pulsation of cortical vessels. BCAS impaired vascular pulsation in pial arteries in WT and Tg-SwDI mice. Our findings show that BCAS influences VCI and that this is paralleled by impaired glymphatic drainage and reduced vascular pulsation. We propose that these additional targets need to be considered when treating VCI

The UNC-45 Chaperone Is Critical for Establishing Myosin-Based Myofibrillar Organization and Cardiac Contractility in the Drosophila Heart Model

UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented

Cyanobacterial lipopolysaccharides and human health – a review

Cyanobacterial lipopolysaccharide/s (LPS) are frequently cited in the cyanobacteria literature as toxins responsible for a variety of heath effects in humans, from skin rashes to gastrointestinal, respiratory and allergic reactions. The attribution of toxic properties to cyanobacterial LPS dates from the 1970s, when it was thought that lipid A, the toxic moiety of LPS, was structurally and functionally conserved across all Gram-negative bacteria. However, more recent research has shown that this is not the case, and lipid A structures are now known to be very different, expressing properties ranging from LPS agonists, through weak endotoxicity to LPS antagonists. Although cyanobacterial LPS is widely cited as a putative toxin, most of the small number of formal research reports describe cyanobacterial LPS as weakly toxic compared to LPS from the Enterobacteriaceae. We systematically reviewed the literature on cyanobacterial LPS, and also examined the much lager body of literature relating to heterotrophic bacterial LPS and the atypical lipid A structures of some photosynthetic bacteria. While the literature on the biological activity of heterotrophic bacterial LPS is overwhelmingly large and therefore difficult to review for the purposes of exclusion, we were unable to find a convincing body of evidence to suggest that heterotrophic bacterial LPS, in the absence of other virulence factors, is responsible for acute gastrointestinal, dermatological or allergic reactions via natural exposure routes in humans. There is a danger that initial speculation about cyanobacterial LPS may evolve into orthodoxy without basis in research findings. No cyanobacterial lipid A structures have been described and published to date, so a recommendation is made that cyanobacteriologists should not continue to attribute such a diverse range of clinical symptoms to cyanobacterial LPS without research confirmation

Over-expression of NCS-1 in AtT-20 cells affects ACTH secretion and storage

The effect of over-expressing neuronal calcium sensor I (NCS-1) upon stimulated adrenocorticotrophin (ACTH) secretion was studied in AtT-20 cells. Stably-transfected AtT-20 cell lines over-expressing NCS-1 were obtained and compared to wild type AtT-20 cells. Corticotrophin releasing factor (CRF-41)-stimulated ACTH secretion from NCS-1 over-expressing cells was significantly reduced from that obtained in wild type AtT-20 cells. The effects of other stimulants of ACTH secretion from wild type AtT-20 cells were not attenuated in NCS-I over-expressing cells. Calcium, guanosine 5'-O-(3'-thiotriphosphate) (GTP-gamma -S) and mastoparan stimulated ACTH secretion from permeabilised wild type AtT-20 and NCS-I over-expressing AtT-20 cells with significantly greater ACTH secretion obtained in NCS-I over-expressing cells. This study shows that in intact cells over-expression of NCS-I reduces exocytotic ACTH release, while in permeabilised cells increases ACTH release. NCS-I has multiple cellular targets and that directly and indirectly via these targets acts to increase the releasable ACTH pool while inhibiting CRF-41 stimulus-secretion coupling. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.</p

Over-expression of NCS-1 in AtT-20 cells affects ACTH secretion and storage

The effect of over-expressing neuronal calcium sensor I (NCS-1) upon stimulated adrenocorticotrophin (ACTH) secretion was studied in AtT-20 cells. Stably-transfected AtT-20 cell lines over-expressing NCS-1 were obtained and compared to wild type AtT-20 cells. Corticotrophin releasing factor (CRF-41)-stimulated ACTH secretion from NCS-1 over-expressing cells was significantly reduced from that obtained in wild type AtT-20 cells. The effects of other stimulants of ACTH secretion from wild type AtT-20 cells were not attenuated in NCS-I over-expressing cells. Calcium, guanosine 5'-O-(3'-thiotriphosphate) (GTP-gamma -S) and mastoparan stimulated ACTH secretion from permeabilised wild type AtT-20 and NCS-I over-expressing AtT-20 cells with significantly greater ACTH secretion obtained in NCS-I over-expressing cells. This study shows that in intact cells over-expression of NCS-I reduces exocytotic ACTH release, while in permeabilised cells increases ACTH release. NCS-I has multiple cellular targets and that directly and indirectly via these targets acts to increase the releasable ACTH pool while inhibiting CRF-41 stimulus-secretion coupling. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.</p