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A zebrafish model for calcineurin-dependent brain function.

ABSTRACT: Small-molecule modulators of calcineurin signaling have been proposed as potential therapeutics in Down syndrome and Alzheimer's disease. Models predict that in Down syndrome, suppressed calcineurin-NFAT signaling may be mitigated by proINDY, which activates NFAT, the nuclear factor of activated T-cells. Conversely, elevated calcineurin signaling in Alzheimer's disease may be suppressed with the calcineurin inhibitors cyclosporine and tacrolimus. Such small-molecule treatments may have both beneficial and adverse effects. The current study examines the effects of proINDY, cyclosporine and tacrolimus on behavior, using zebrafish larvae as a model system. To suppress calcineurin signaling, larvae were treated with cyclosporine and tacrolimus. We found that these calcineurin inhibitors induced hyperactivity, suppressed visually-guided behaviors, acoustic hyperexcitability and reduced habituation to acoustic stimuli. To activate calcineurin-NFAT signaling, larvae were treated with proINDY. ProINDY treatment reduced activity and stimulated visually-guided behaviors, opposite to the behavioral changes induced by calcineurin inhibitors. The opposing effects suggest that activity and visually-guided behaviors are regulated by the calcineurin-NFAT signaling pathway. A central role of calcineurin-NFAT signaling is further supported by co-treatments of calcineurin inhibitors and proINDY, which had therapeutic effects on activity and visually-guided behaviors. However, these co-treatments adversely increased excitability, suggesting that some behaviors are regulated by other calcineurin signaling pathways. Overall, the developed methodologies provide an efficient high-throughput platform for the evaluation of modulators of calcineurin signaling that restore neural function, while avoiding adverse side effects, in a complex neural system.

SUBMITTER: Tucker Edmister S

PROVIDER: S-EPMC8903086 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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A zebrafish model for calcineurin-dependent brain function.

Tucker Edmister Sara S Ibrahim Rahma R Kakodkar Rohit R Kreiling Jill A JA Creton Robbert R

Behavioural brain research 20210820

Small-molecule modulators of calcineurin signaling have been proposed as potential therapeutics in Down syndrome and Alzheimer's disease. Models predict that in Down syndrome, suppressed calcineurin-NFAT signaling may be mitigated by proINDY, which activates NFAT, the nuclear factor of activated T-cells. Conversely, elevated calcineurin signaling in Alzheimer's disease may be suppressed with the calcineurin inhibitors cyclosporine and tacrolimus. Such small-molecule treatments may have both bene ...[more]

PMID: 34425181

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Project description:Arctigenin, a lignan-derived compound, is a constituent of the seeds of Arctium lappa. Arctigenin was previously shown to inhibit osteoclastogenesis; however, this inhibitory mechanism has yet to be elucidated. Here, we showed that arctigenin inhibited the action of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a key transcription factor for osteoclastogenesis. NFATc1 in osteoclast precursors was activated through two distinct pathways: the calcineurin-dependent and osteoblastic cell-dependent pathways. Among the several lignan-derived compounds examined, arctigenin most strongly inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclast-like cell formation in mouse bone marrow macrophage (BMM) cultures, in which the calcineurin-dependent NFATc1 pathway was activated. Arctigenin suppressed neither the activation of nuclear factor κB and mitogen-activated protein kinases nor the up-regulation of c-Fos expression in BMMs treated with RANKL. However, arctigenin suppressed RANKL-induced NFATc1 expression. Interestingly, the treatment of osteoclast-like cells with arctigenin converted NFATc1 into a lower molecular weight species, which was translocated into the nucleus even in the absence of RANKL. Nevertheless, arctigenin as well as cyclosporin A (CsA), a calcineurin inhibitor, suppressed the NFAT-luciferase reporter activity induced by ionomycin and phorbol 12-myristate 13-acetate in BMMs. Chromatin immunoprecipitation analysis confirmed that arctigenin inhibited the recruitment of NFATc1 to the promoter region of the NFATc1 target gene. Arctigenin, but not CsA suppressed osteoclast-like cell formation in co-cultures of osteoblastic cells and bone marrow cells, in which the osteoblastic cell-dependent NFATc1 pathway was activated. The forced expression of constitutively active NFATc1 rescued osteoclastogenesis in BMM cultures treated with CsA, but not that treated with arctigenin. Arctigenin also suppressed the pit-forming activity of osteoclast-like cells cultured on dentin slices. These results suggest that arctigenin induces a dominant negative species of NFATc1, which inhibits osteoclast differentiation and function by suppressing both calcineurin-dependent and osteoblastic cell-dependent NFATc1 pathways.

Project description:Prolonged hyperglycemia causes long-term vision complications and an increased risk of cognitive deficits. High blood sugar also confers an osmotic load/stress to cells. We assessed behavioral and neurochemical changes in zebrafish brain and retina following prolonged hyperglycemia for 4-weeks or 8-weeks. At each time point, behavior was assessed using 3-chamber choice task and optomotor response; tissue was then collected and levels of inflammatory markers, tight junction proteins, and neurotransmitters determined using Western Blots. After 4-weeks, brain levels of v-rel reticuloendotheliosis viral oncogene homolog A (avian) (RelA; NF-kB subunit), IkB kinase (IKK), and glial fibrillary acidic protein (GFAP) were significantly elevated; differences in zonula occludens-1 (ZO-1), claudin-5, glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) were not significant. In retina, significant differences were observed only for TH (decreased), Rel A (increased), and GFAP (increased) levels. Glucose-specific differences in initial choice latency and discrimination ratios were also observed. After 8-weeks, RelA, GAD, and TH were significantly elevated in both tissues; IKK and GFAP levels were also elevated, though not significantly. ZO-1 and claudin-5 levels osmotically decreased in retina but displayed an increasing trend in glucose-treated brains. Differences in discrimination ratio were driven by osmotic load. OMRs increased in glucose-treated fish at both ages. In vivo analysis of retinal vasculature suggested thicker vessels after 4-weeks, but thinner vessels at 8-weeks. In vitro, glucose treatment reduced formation of nodes and meshes in 3B-11 endothelial cells, suggesting a reduced ability to form a vascular network. Overall, hyperglycemia triggered a strong inflammatory response causing initial trending changes in tight junction and neuronal markers. Most differences after 4-weeks of exposure were observed in glucose-treated fish suggesting effects on glucose metabolism independent of osmotic load. After 8-weeks, the inflammatory response remained and glucose-specific effects on neurotransmitter markers were observed. Osmotic differences impacted cognitive behavior and retinal protein levels; protein levels in brain displayed glucose-driven changes. Thus, we not only observed differential sensitivities of retina and brain to glucose-insult, but also different cellular responses, suggesting hyperglycemia causes complex effects at the cellular level and/or that zebrafish are able to compensate for the continued high blood glucose levels.

Project description:RationaleStudies to dissect the role of calcineurin in pathological cardiac remodeling have relied heavily on murine models, in which genetic gain- and loss-of-function manipulations are initiated at or before birth. However, the great majority of clinical cardiac pathology occurs in adults. Yet nothing is known about the effects of calcineurin when its activation commences in adulthood. Furthermore, despite the fact that ventricular hypertrophy is a well-established risk factor for heart failure, the relative pace and progression of these 2 major phenotypic features of heart disease are unknown. Finally, even though therapeutic interventions in adults are designed to slow, arrest, or reverse disease pathogenesis, little is known about the capacity for spontaneous reversibility of calcineurin-dependent pathological remodeling.ObjectiveWe set out to address these 3 questions by studying mice engineered to harbor in cardiomyocytes a constitutively active calcineurin transgene driven by a tetracycline-responsive promoter element.Methods and resultsExpression of the mutant calcineurin transgene was initiated for variable lengths of time to determine the natural history of disease pathogenesis, and to determine when, if ever, these events are reversible. Activation of the calcineurin transgene in adult mice triggered rapid and robust cardiac growth with features characteristic of pathological hypertrophy. Concentric hypertrophy preceded the development of systolic dysfunction, fetal gene activation, fibrosis, and clinical heart failure. Furthermore, cardiac hypertrophy reversed spontaneously when calcineurin activity was turned off, and expression of fetal genes reverted to baseline. Fibrosis, a prominent feature of pathological cardiac remodeling, manifested partial reversibility.ConclusionsTogether, these data establish and define the deleterious effects of calcineurin signaling in the adult heart and reveal that calcineurin-dependent hypertrophy with concentric geometry precedes systolic dysfunction and heart failure. Furthermore, these findings demonstrate that during much of the disease process, calcineurin-dependent remodeling remains reversible.

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A zebrafish model for calcineurin-dependent brain function.

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A zebrafish model for calcineurin-dependent brain function.

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