Luiza Wilges Kist

Microcystin-LR acute exposure increases AChE activity via transcriptional ache activation in zebrafish (Danio rerio) brain

Microcystins (MCs) constitute a family of cyanobacterial toxins, with more than 80 variants. These toxins are able to induce hepatotoxicity in several organisms mainly through the inhibition of protein phosphatases PP1 and PP2A and oxidative stress generation. Since recent evidence shows that MCs can either accumulate in brain or alter behavior patterns of fish species, in this study we tested the in vitro and in vivo effects of MC-LR at different concentrations on acetylcholinesterase (AChE) activity in zebrafish brain. In vivo studies showed that 100 μg/L MC-LR led to a significant increase in the AChE activity (27%) when zebrafish were exposed to the toxin dissolved in water, but did not cause any significant changes when injected intraperitoneally. In addition, semiquantitative RT-PCR analysis demonstrated that 100 μg/L MC-LR exposure also increased ache mRNA levels in zebrafish brain. The in vitro assays did not reveal any significant changes in AChE activity. These findings provide the first evidence that brain AChE is another potential target for MCs and suggest that the observed increases in AChE enzymatic activity and in ache transcript levels after MC-LR exposure depend, at least partially, on branchial uptake or ingestion.

Persistent impaired glucose metabolism in a zebrafish hyperglycemia model

Diabetes mellitus (DM) affects over 10% of the worlds population. Hyperglycemia is the main feature for the diagnosis of this disease. The zebrafish (Danio rerio) is an established model organism for the study of various metabolic diseases. In this paper, hyperglycemic zebrafish, when immersed in a 111 mM glucose solution for 14 days, developed increased glycation of proteins from the eyes, decreased mRNA levels of insulin receptors in the muscle, and a reversion of high blood glucose level after treatment with anti-diabetic drugs (glimepiride and metformin) even after 7 days of glucose withdrawal. Additionally, hyperglycemic zebrafish developed an impaired response to exogenous insulin, which was recovered after 7 days of glucose withdrawal. These data suggest that the exposure of adult zebrafish to high glucose concentration is able to induce persistent metabolic changes probably underlined by a hyperinsulinemic state and impaired peripheral glucose metabolism.

Prevention of unpredictable chronic stress-related phenomena in zebrafish exposed to bromazepam, fluoxetine and nortriptyline

RationaleSeveral model organisms have been employed to study the impacts of stress on biological systems. Different models of unpredictable chronic stress (UCS) have been established in rodents; however, these protocols are expensive, long-lasting, and require a large physical structure. Our group has recently reported an UCS protocol in zebrafish with several advantages compared to rodent models. We observed that UCS induced behavioral, biochemical, and molecular changes similar to those observed in depressed patients, supporting the translational relevance of the protocol.ObjectivesConsidering that a pharmacological assessment is lacking in this zebrafish model, our aim was to evaluate the effects of anxiolytic (bromazepam) and antidepressant drugs (fluoxetine and nortriptyline) on behavioral (novel tank test), biochemical (whole-body cortisol), and molecular parameters (cox-2, tnf-α, il-6, and il-10 gene expression) in zebrafish subjected to UCS.ResultsWe replicated previous data showing that UCS induces behavioral and neuroendocrine alterations in zebrafish, and we show for the first time that anxiolytic and antidepressant drugs are able to prevent such effects. Furthermore, we extended the molecular characterization of the model, revealing that UCS increases expression of the pro-inflammatory markers cox-2 and il-6, which was also prevented by the drugs tested.ConclusionsThis study reinforces the use of zebrafish as a model organism to study the behavioral and physiological effects of stress. The UCS protocol may also serve as a screening tool for evaluating new drugs that can be used to treat psychiatric disorders with stress-related etiologies.

Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio).

Diabetes mellitus, which causes hyperglycemia, affects the central nervous system and can impairs cognitive functions, such as memory. The aim of this study was to investigate the effects of hyperglycemia on memory as well as on the activity of acethylcholinesterase. Hyperglycemia was induced in adult zebrafish by immersion in glucose 111mM by 14 days. The animals were divided in 4 groups: control, glucose-treated, glucose-washout 7-days and glucose-washout 14-days. We evaluated the performance in inhibitory avoidance task and locomotor activity. We also determined acethylcholinesterase activity and gene expression from whole brain. In order to counteract the effect of hyperglycemia underlined by effects on acethylcholinesterase activity, we treated the animals with galantamine (0.05ng/g), an inhibitor of this enzyme. Also we evaluated the gene expression of insulin receptor and glucose transporter from zebrafish brain. The hyperglycemia promoted memory deficit in adult zebrafish, which can be explained by increased AChE activity. The ache mRNA levels from zebrafish brain were decrease in 111mM glucose group and returned to normal levels after 7 days of glucose withdrawal. Insulin receptors (insra-1, insra-2, insrb-1 and insrb-2) and glut-3 mRNA levels were not significantly changed. Our results also demonstrated that galantamine was able to reverse the memory deficit caused by hyperglycemia, demonstrating that these effects involve modulation of AChE activity. These data suggest that the memory impairment induced by hyperglycemia is underlined by the cholinergic dysfunction caused by the mechanisms involving the control of acetylcholinesterase function and gene expression.

L-tyrosine administration increases acetylcholinesterase activity in rats.

Tyrosinemia is a rare genetic disease caused by mutations on genes that codify enzymes responsible for tyrosine metabolism. Considering that tyrosinemics patients usually present symptoms associated with central nervous system alterations that ranges from slight decreases in intelligence to severe mental retardation, we decided to investigate whether acute and chronic administration of L-tyrosine in rats would affect acetylcholinesterase mRNA expression and enzymatic activity during their development. In our acute protocol, Wistar rats (10 and 30 days old) were killed one hour after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. Chronic administration consisted of L-tyrosine (500 mg/kg) or saline injections 12 h apart for 24 days in Wistar rats (7 days old) and rats were killed 12 h after last injection. Acetylcholinesterase activity was measured by Ellmans method and acetylcholinesterase expression was carried out by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) assay. We observed that acute (10 and 30 days old rats) and chronic L-tyrosine administration increased acetylcholinesterase activity in serum and all tested brain areas (hippocampus, striatum and cerebral cortex) when compared to control group. Moreover, there was a significant decrease in mRNA levels of acetylcholinesterase in hippocampus was observed after acute protocol (10 and 30 days old rats) and in striatum after chronic protocol. In case these alterations also occur in the brain of the patients, our results may explain, at least in part, the neurological sequelae associated with high plasma concentrations of tyrosine seen in patients affected by tyrosinemia type II.

Acute and Chronic Administration of the Branched-Chain Amino Acids Decreases Nerve Growth Factor in Rat Hippocampus

Maple syrup urine disease (MSUD) is a neurometabolic disorder caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase leading to accumulation of the branched-chain amino acids (BCAA) and their corresponding branched-chain α-keto acids. In this study, we examined the effects of acute and chronic administration of BCAA on protein levels and mRNA expression of nerve growth factor (NGF) considering that patients with MSUD present neurological dysfunction and cognitive impairment. Considering previous observations, it is suggested that oxidative stress may be involved in the pathophysiology of the neurological dysfunction of MSUD. We also investigated the influence of antioxidant treatment (N-acetylcysteine and deferoxamine) in order to verify the influence of oxidative stress in the modulation of NGF levels. Our results demonstrated decreased protein levels of NGF in the hippocampus after acute and chronic administration of BCAA. In addition, we showed a significant decrease in the expression of ngf in the hippocampus only following acute administration in 10-day-old rats. Interestingly, antioxidant treatment was able to prevent the decrease in NGF levels by increasing ngf expression. In conclusion, the results suggest that BCAA is involved in the regulation of NGF in the developing rat. Thus, it is possible that alteration of neurotrophin levels during brain maturation could be of pivotal importance in the impairment of cognition provoked by BCAA. Moreover, the decrease in NGF levels was prevented by antioxidant treatment, reinforcing that the hypothesis of oxidative stress can be an important pathophysiological mechanism underlying the brain damage observed in MSUD.

Evaluation of Acetylcholinesterase in an Animal Model of Maple Syrup Urine Disease

Maple syrup urine disease is an inherited metabolic disease predominantly characterized by neurological dysfunction. However, the mechanisms underlying the neuropathology of this disease are still not defined. Therefore, the aim of this study was to investigate the effect of acute and chronic administration of a branched-chain amino acids (BCAA) pool (leucine, isoleucine, and valine) on acetylcholinesterase (AChE) activity and gene expression in the brain and serum of rats and to assess if antioxidant treatment prevented the alterations induced by BCAA administration. Our results show that the acute administration of a BCAA pool in 10- and 30-day-old rats increases AChE activity in the cerebral cortex, striatum, hippocampus, and serum. Moreover, chronic administration of the BCAA pool also increases AChE activity in the structures studied, and antioxidant treatment prevents this increase. In addition, we show a significant decrease in the mRNA expression of AChE in the hippocampus following acute administration in 10- and 30-day-old rats. On the other hand, AChE expression increased significantly after chronic administration of the BCAA pool. Interestingly, the antioxidant treatment was able to prevent the increased AChE activity without altering AChE expression. In conclusion, the results from the present study demonstrate a marked increase in AChE activity in all brain structures following the administration of a BCAA pool. Moreover, the increased AChE activity is prevented by the coadministration of N-acetylcysteine and deferoxamine as antioxidants.

Reduction of Na + ,K + -ATPase activity and expression in cerebral cortex of glutaryl-CoA dehydrogenase deficient mice: A possible mechanism for brain injury in glutaric aciduria type I

Mitochondrial dysfunction has been proposed to play an important role in the neuropathology of glutaric acidemia type I (GA I). However, the relevance of bioenergetics disruption and the exact mechanisms responsible for the cortical leukodystrophy and the striatum degeneration presented by GA I patients are not yet fully understood. Therefore, in the present work we measured the respiratory chain complexes activities I-IV, mitochondrial respiratory parameters state 3, state 4, the respiratory control ratio and dinitrophenol (DNP)-stimulated respiration (uncoupled state), as well as the activities of α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and Na+, K+-ATPase in cerebral cortex, striatum and hippocampus from 30-day-old Gcdh-/- and wild type (WT) mice fed with a normal or a high Lys (4.7%) diet. When a baseline (0.9% Lys) diet was given, we verified mild alterations of the activities of some respiratory chain complexes in cerebral cortex and hippocampus, but not in striatum from Gcdh-/- mice as compared to WT animals. Furthermore, the mitochondrial respiratory parameters and the activities of α-KGDH and CK were not modified in all brain structures from Gcdh-/- mice. In contrast, we found a significant reduction of Na(+), K(+)-ATPase activity associated with a lower degree of its expression in cerebral cortex from Gcdh-/- mice. Furthermore, a high Lys (4.7%) diet did not accentuate the biochemical alterations observed in Gcdh-/- mice fed with a normal diet. Since Na(+), K(+)-ATPase activity is required for cell volume regulation and to maintain the membrane potential necessary for a normal neurotransmission, it is presumed that reduction of this enzyme activity may represent a potential underlying mechanism involved in the brain swelling and cortical abnormalities (cortical atrophy with leukodystrophy) observed in patients affected by GA I.

Maternal caffeine exposure alters neuromotor development and hippocampus acetylcholinesterase activity in rat offspring

The objective of this study was to evaluate the effects of maternal caffeine intake on the neuromotor development of rat offspring and on acetylcholine degradation and acetylcholinesterase (AChE) expression in the hippocampus of 14-day-old infant rats. Rat dams were treated with caffeine (0.3g/L) throughout gestation and lactation until the pups were 14 days old. The pups were divided into three groups: (1) control, (2) caffeine, and (3) washout caffeine. The washout group received a caffeine solution until the seventh postnatal day (P7). Righting reflex (RR) and negative geotaxis (NG) were assessed to evaluate postural parameters as an index of neuromotor reflexes. An open-field (OF) test was conducted to assess locomotor and exploratory activities as well as anxiety-like behaviors. Caffeine treatment increased both RR and NG latency times. In the OF test, the caffeine group had fewer outer crossings and reduced locomotion compared to control, while the washout group showed increased inner crossings in relation to the other groups and fewer rearings only in comparison to the control group. We found decreased AChE activity in the caffeine group compared to the other groups, with no alteration in AChE transcriptional regulation. Chronic maternal exposure to caffeine promotes important alterations in neuromotor development. These results highlight the ability of maternal caffeine intake to interfere with cholinergic neurotransmission during brain development.

Acute Exposure to Microcystin-Producing Cyanobacterium Microcystis aeruginosa Alters Adult Zebrafish (Danio rerio) Swimming Performance Parameters

Microcystins (MCs) are toxins produced by cyanobacteria (blue-green algae), primarily Microcystis aeruginosa, forming water blooms worldwide. When an organism is exposed to environmental perturbations, alterations in normal behavioral patterns occur. Behavioral repertoire represents the consequence of a diversity of physiological and biochemical alterations. In this study, we assessed behavioral patterns and whole-body cortisol levels of adult zebrafish (Danio rerio) exposed to cell culture of the microcystin-producing cyanobacterium M. aeruginosa (MC-LR, strain RST9501). MC-LR exposure (100 μg/L) decreased by 63% the distance traveled and increased threefold the immobility time when compared to the control group. Interestingly, no significant alterations in the number of line crossings were found at the same MC-LR concentration and time of exposure. When animals were exposed to 50 and 100 μg/L, MC-LR promoted a significant increase (around 93%) in the time spent in the bottom portion of the tank, suggesting an anxiogenic effect. The results also showed that none of the MC-LR concentrations tested promoted significant alterations in absolute turn angle, path efficiency, social behavior, or whole-body cortisol level. These findings indicate that behavior is susceptible to MC-LR exposure and provide evidence for a better understanding of the ecological consequences of toxic algal blooms.