Esculentin-2CHa (GA30) mitigates copper-induced redox imbalance and behavioural deficit in Drosophila melanogaster

Esculentin-2CHa (GA30) mitigates copper-induced redox imbalance and behavioural deficit in Drosophila melanogaster | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Esculentin-2CHa (GA30) mitigates copper-induced redox imbalance and behavioural deficit in Drosophila melanogaster Onyedika L. Udochukwu, Aghogho Oyibo, Ayodele A. Falobi, Amos O. Abolaji, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4368804/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Excess copper ion (Cu 2+ ) has been implicated in various pathological conditions involving oxidative stress and inflammation. This study investigated neuroprotective effects of esculentin-2CHa-(GA30) on copper-induced toxicity in Drosophila melanogaster . Flies were treated with esculentin-2CHa (5.0 and 7.5 µM/kg diet) and/or Cu 2+ (1mM) orally for 5 days. Effects of esculetin-2CHa-(GA30) on markers of redox-antioxidant status and neuro-behavioural activities were assessed. Esculetin-2CHa-(GA30) did not affect survival rate but reversed the effect of copper on eclosion rate. Esculetin-2CHa-(GA30) dose-dependently mitigated Cu 2+ -induced elevation of hydrogen peroxide (15.1–15.8%, P < 0.05), thiobarbituric reactive substance (37.2–55.1%, P < 0.01–0.001) and protein carbonyl (20.7–63.8%, P < 0.05–0.001). Esculetin-2CHa-(GA30) ameliorated Cu 2+ -induced inhibition of catalase (1.5–1.7-fold, P < 0.01–0.001), glutathione S-transferase activities (1.5–2.1-fold, P < 0.01–0.001) and decline in non-protein thiols levels (13.6–27.7%, P < 0.05). Esculetin-2CHa-(GA30) reduced Cu 2+− induced elevation of monoamine oxidase (21.7–39.7%, P < 0.05–0.01) and acetylcholinesterase (40.1–55.9%, P < 0.01–0.001) activities. Copper-induced impaired locomotor activities were dose-dependently improved in esculentin-2CH-(GA30)-treated flies (21.4%, P < 0.05 and 72.1%, P < 0.01). Histological assessments indicated the ability of esculentin-2CHa-(GA30) to sequester Cu 2+ in the microglia. In conclusion, esculentin-2CHa-(GA30) exhibited its neuroprotective effects through improved balance of redox status and associated behavioural characteristics. Further studies to delineate molecular mechanisms underlying observed effects would be required. Biological sciences/Biochemistry Biological sciences/Developmental biology Biological sciences/Drug discovery Biological sciences/Neuroscience Health sciences/Diseases Copper toxicity esculentin-2CHa-(GA30) amphibian skin peptides neurodegeneration neuroprotective effects Drosophila melanogaster INTRODUCTION Copper (Cu) is a transition metal that is essential for many cellular processes 1 . It facilitates several physiological processes including antioxidant defense, collagen synthesis, maintenance of blood vessel integrity, oxygen metabolism, skin pigmentation, synthesis of neurotransmitters, and the regulation of iron homeostasis 2 . However, emerging evidence indicates that the redox activity that makes copper physiologically essential is deleterious at excessive cellular levels of copper ions 3,4 . Aberration in copper homeostasis may result from aging, environmental influences, or genetic mutation and this has been implicated in a variety of abnormal physiological conditions like cancer, inflammation, and neurodegeneration 5,6,7 . Recently, the pathology of Alzheimer’s disease (AD) has been traced to the imbalances of intracellular copper homeostasis and it has been shown that exposure to high copper concentration could lead to increased Amyloid Precursor Protein (APP) expression, amyloid beta (Aβ) aggregation, and hyperphosphorylation of tau 8 . Similarly, previous studies revealed that excessive copper intake affects cognition and plays a role in the pathogenesis of AD, Parkinson Disease, and amyotrophic lateral sclerosis 9 . Cruces-Sande et al. 10 assessed in vivo effects of copper in brain oxidative damage and its ability to increase dopaminergic degeneration induced by 6-hydroxydopamine. The study reported that chronic copper administration caused the accumulation of 6-hydroxydopamine in different areas of the brain (cortex, striatum, nigra). The accumulation of 6-hydroxydopamine was also observed to be associated with increased levels of Thiobarbituric Reactive Substance (TBARS), decreased levels of protein free-thiol in the cortex, reduced catalase activity and increased in glutathione peroxidase activity 10 . This indicates that copper potentiates the action of other factors involved in the pathogenesis of neurodegenerative diseases through oxidative stress. The quest to preserve copper homeostasis opened up new therapeutic avenues in the definition of the novel disease-modifying bioactive peptide, esculentin-2CHa(GA30) which has a dearth of information on its neuroprotective potential. Esculentin-2CHa (GFSSIFRGVAKFASKGLGKDLAKLGVDLVA) is the truncated version of esculentin-2CHa (which has 37 amino acid residues) originally isolated from the skin secretion of the Chiricahua leopard frog, Lithobates chiricahuensis 11 . The frog skin host-defense peptide, esculentin-2CHa has been shown to display multifunctional effects including antimicrobial, antitumor, and immunomodulatory properties 11 . Esculentin-2CHa(GA30) is the truncated version of the original peptide, lacking the last seven amino acid residues 12 . Antidiabetic 12 and antioxidative effects 13 of esculentin-2CHa(GA30) have been reported. These previously reported biological effects motivated the selection of esculentin-2CHa(GA30) and the hypothesis that esculentin-2CHa( 30 ) will elicit neuroprotective effects against copper toxicity. Although most studies involving metal toxicity and neurodegeneration use vertebrate models (humans/ rodents). However, invertebrate models are now been considered as alternative useful and cheaper models. In this study, Drosophila melanogaster was selected as a model organism due to its extensive metabolic and genetic similarity to humans 14 . It has been reported that D. melanogaster is about 60% genetically similar to humans and has several similar biological mechanisms 9,14 . In this study, we investigated neuroprotective effects as well as the ability of esculentin-2CHa(GA30) to protect D. melanogaster against oxidative damage and neuro-behavioural deficit associated with copper-induced toxicity. RESULTS Effect of esculentin-2CHa(GA30) on survival and eclosion rates in Drosophila melanogaster The rate at which control flies as well as flies treated with Cu 2+ in the absence or presence of esculentin-2CHa(GA30) die over a 5-day period is presented in Figs. 1 A and 1 B. Data presented in these Figures indicate a similar death rate in both groups. However, there are differences in the number of surviving flies across the groups on Day 5. Specifically, 26.6 ± 0.5 flies in the control group survived up to Day 5 (Fig. 1 C). This is similar to the average number of surviving flies in groups treated with the peptide at 5.0µM/kg diet (25.4 ± 0.9 flies) and 7.5µM/kg diet (27.2 ± 1.1 flies). The treatment of flies with Cu 2+ reduced the number of surviving flies by 14.3% (P < 0.05). This reduction was prevented by treated of flies with the peptide at both 5.0µM/kg diet (25.2 ± 1.3 flies) and 7.5µM/kg diet (24.8 ± 1.0 flies). The impact of the exposure of flies to Cu 2+ on eclosion rates was more pronounced (Fig. 1 D and 1 E). The rate of emergence of new flies in Cu 2+ treated groups reduced by 34.8% (P < 0.01) compared to the control group. Eclosion rate in the group treated with the peptide at 5.0µM/kg diet was similar to the control group. At the peptide concentration of 7.5µM/kg diet, eclosion rate increased by 1.6-fold (P < 0.01) in the absence of Cu 2+ and by 1.1-fold (ns) in the presence of Cu 2+ compared to control flies. The effect observed in the group treated with peptide (7.5µM/kg diet) and Cu 2+ represent a 1.7-fold (P < 0.05) increase compared to flies treated with Cu 2+ alone. With respect to the number of emerging flies on Day 12, a similar number of emerging flies was observed in the control and the Cu 2+ groups. The number of emerging flies in the group treated with 5.0µM/kg diet alone is also similar to the number observed in the control group and represent a 12.3% (ns) increase compared to the number of emerging flies observed in the group treated with Cu 2+ at the same peptide concentration. Compared to all other groups, the number of emerging flies was significantly higher in the group treated with the peptide alone at the dose of 7.5µM/kg diet (1.5-fold, P < 0.01). The effect of Cu 2+ on the number of emerging flies was also completely inhibited at this peptide concentration (Fig. 1 F). Effects of esculentin-2CHa(GA30) on H 2 O 2 and NO levels in Cu 2+ -treated Drosophila melanogaster H 2 O 2 and NO levels were assessed as markers of oxidative stress in this study. Results obtained indicate that treatment of flies with Cu 2+ led to 16.2% (P < 0.05 increase in H 2 O 2 levels (Fig. 2 A) compared to control flies. Unlike Cu 2+ , the treatment of flies with esculentin-2CHa(GA30) alone at all concentrations tested did not produce significant increase in H 2 O 2 levels. However, the elevation of H 2 O 2 levels produced by Cu 2+ was reduced by 15.8% (P < 0.05) and 15.1% (P < 0.05) in flies treated with 5.0 and 7.5 µM/kg diet of esculentin-2CHa(GA30) respectively (Fig. 2 A). NO levels in control flies and flies treated with esculentin-2CHa(GA30) were similar. However, treated with Cu 2+ increased NO levels by 36.4% (P < 0.05). The combination of Cu 2+ with esculentin-2CHa(GA30) did not reduce the elevation in NO levels produced by Cu 2+ (Fig. 2 B). Effects of esculentin-2CHa(GA30) on lipid peroxidation and protein carbonylation in Cu 2+ -treated Drosophila melanogaster The concentration of thiobarbituric reactive substances (TBARS) in control flies was observed as 36.5 ± 2.6 nmol/mg protein. The concentration of TBARS was increased by 32.8% (P < 0.01) in flies exposed to Cu 2+ only (Fig. 3 A). The treatment of flies with esculentin-2CHa(GA30) at both dosages used in this study significantly reduced basal and Cu 2+ -induced accumulation of TBARS. Specifically, TBARS in flies treated with the peptide alone at 5.0µM/kg diet was 17.3% (P < 0.05) lower compared to control flies. The peptide at 5.0µM/kg diet reduced Cu 2+ -induced elevation of TBARS by 37.2% (P < 0.01). A more significant reduction in basal (40.8%, P < 0.01) and Cu 2+ -induced (55.1%, P < 0.001) TBARS levels was observed in flies treated with esculentin-2CHa(GA30) at 7.5µM/kg diet (Fig. 3 A). Similarly, protein carbonyl levels in Cu 2+ -treated flies in the absence of the peptide increased by 1.5-fold (P < 0.01) compared with control flies. However, the treatment of flies with esculentin-2CHa(GA30) at 5.0µM/kg diet reduced basal (20.7%, P < 0.05) and Cu 2+ -induced (47.0%, P < 0.01) levels of protein carbonyls. Similarly, reduced basal (33.6%, P < 0.01) and Cu 2+ -induced (63.8%, P < 0.001) levels of protein carbonyls were observed in flies treated with the peptide at 7.5µM/kg diet (Fig. 3 B). Effects of Esculentin-2CHa(GA30) on activities of catalase and glutathione-S-transferase in Cu 2+ -treated Drosophila melanogaster Basal catalase activities in control flies were estimated as 90.2 ± 3.3 nmol/mg protein. This was significantly reduced by the exposure of flies to Cu2+ (74.0%, P < 0.001, Fig. 4 A). In the presence of peptide alone at 5.0µM/kg diet, a reduction of 28.6% (P < 0.05) in catalase activities was also observed. However, the combination of esculentin-2CHa(GA30) (5.0µM/kg diet) with Cu 2+ resulted in the inhibition of Cu 2+ -induced reduction in catalase activity (55.7%, P < 0.01). At peptide concentration of 7.5µM/kg diet alone, an increase of 1.7-fold (P < 0.001) was observed compared with control flies. When the peptide at 7.5µM/kg diet was combined with Cu2+, the deleterious effects of Cu2 + on catalase activities was completely reversed (Fig. 4 A). Similarly, glutathione-s-transferase activities in control flies were estimated as 0.85 ± 0.06 nmol/kg protein. These activities were reduced by 52.7% (P < 0.01) in flies exposed to Cu2+. Treatment with esculentin-2CHa(GA30) alone increased glutathione-s-transferase activities by 1.6-fold (P < 0.01) and 2.1-fold (P < 0.001) at peptide concentrations of 5.0 and 7.5 µM/kg diet respectively. In the presence of Cu2+, the peptide inhibited the deleterious effects of Cu2 + by 67.2% (P < 0.001) and 86.6% (P < 0.001) (Fig. 4 B). Effects of esculentin-2CHa(GA30) on levels of non-protein thiols and total thiols in Cu 2+ -treated Drosophila melanogaster The treatment of flies with esculentin-2CHa(GA30) at 5.0µM/kg diet did not affect the level of non-protein thiols when compared with control flies (Fig. 4 C). A similar result was obtained at the peptide concentration of 7.5µM/kg diet. However, the level of non-protein thiols in flies treated with Cu 2+ was reduced by 27.7% (P < 0.05). Esculentin-2CHa(GA30) at 5.0µM/kg diet increased the level of non-protein thiols by 13.6% (ns) compared to Cu 2+ -treated flies and at a peptide concentration of 7.5µM/kg diet, the deleterious effect of Cu 2+ was completely inhibited (Fig. 4 D). The treatment of flies with esculentin-2CHa(GA30) at 5.0 and 7.5 µM/kg diet increased total thiols levels by 26.0% (P < 0.05) and 23.3% (P < 0.05) when compared with control flies (Fig. 4 D). The treatment of flies with Cu2 + also reduced total thiol concentration by 21.2% (P < 0.05). The peptide failed to completely inhibit the effect of Cu 2+ , even though total thiol levels increased by 1.2-fold (P < 0.05) and 1.3-fold (P < 0.05) in flies treated with the peptide at 5.0 and 7.5 µM/kg diet respectively. Effect of esculentin-2CHa(GA30) on behavioural and neurotransmission markers in Drosophila melanogaster Negative geotaxis was observed in 74.7 ± 4.4% of control flies. The treatment of flies with esculentin-2CHa(GA30) did not significantly change the number of flies with the ability for negative geotaxis (Fig. 5 A). However, following the exposure of flies to Cu 2+ , only 52.7 ± 4.1% of flies had the ability for negative geotaxis. Treatment of flies exposed to Cu 2+ with esculentin-2CHa(GA30) at 5.0µM/kg diet completely prevented the effect of Cu 2+ on negative geotaxis. At peptide concentration of 7.5µM/kg diet, the number of flies with the ability for negative geotaxis increased by 21.4% (P < 0.05) compared to control flies and by 72.1% (P < 0.001) compared to untreated flies exposed to Cu 2+ alone (Fig. 5 A). Monoamine oxidase activities in control flies was estimated as 1.3 ± 0.1 nmol/mg protein. These activities were reduced by 13.6% (ns) and 41.5% (P < 0.001) compared to control flies. The exposure of flies to Cu 2+ , increased monoamine oxidase activities by 1.4-fold (P < 0.001). However, the treatment of flies exposed to Cu 2+ with esculentin-2CHa(GA30) at 5.0 and 7.5 µM/kg diet completely inhibited the effect of Cu 2+ and reduced monoamine oxidase by 21.7% (P < 0.05) and 39.7% (P < 0.01) respectively when compared to control flies (Fig. 5 B). Similar effects were observed for acetylcholine esterase activities. Specifically, similar acetylcholine esterase activities were observed in control flies and flies treated with esculentin-2CHa(GA30) at 5.0 and 7.5 µM/kg diet alone (Fig. 5 C). Increased enzyme activities (1.9-fold, P < 0.001) was observed in flies exposed to Cu 2+ . The treatment of Cu 2+ -exposed flies with esculentin-2CHa(GA30) reduced the effect of Cu 2+ by 40.1% (P < 0.01) and 55.9% (P < 0.001) at 5.0 and 7.5 µM/kg diet peptide concentrations respectively. Copper-chelating effects of esculentin-2CHa(GA30) in the brain of Drosophila melanogaster The brain photomicrograph taken across the treatment groups (Fig. 6 ) indicated an even distribution of the neurons around the white matter of control flies and flies treated with esculentin-2CHa(GA30) at all concentrations tested. However, there is atrophy of neurons and white matter in the brain of flies exposed to Cu 2+ . A moderate and significant repopulation of neurons in the grey matter of the lobular of flies co-treated with the peptide and Cu 2+ 5.0µM and 7.5 µM respectively was observed was observed (Fig. 6 ). DISCUSSION Copper is an essential metabolic trace element in all organisms, and it serves as cofactors for many enzymatic reactions 15,16 . However, deleterious effects of excessive copper levels have been reported 9 . The ease of transition between the mono- and the di-ionic states of copper, resulting in the generation of free radicals and oxidative stress, has been implicated in the toxic effects of copper 15,17 . Moreso, imbalance in copper homeostasis has been associated with neurodegeneration due to its ability to bind to hyperphosphorylated tau proteins in neurons 18 . This prescribes a role for imbalance in copper homeostasis in the pathogenesis of diseases such as Alzheimer’s and Parkinson’s diseases. The fact that Drosophila melanogaster possesses unique characteristics such as ease of handling, short life span, genetic tractability, complex behaviors, and well-known simple neuroanatomy made it a good model for neurological studies, such as the one reported in this article 19,20 . The neurotoxic effect of copper in D. melanogaster has been reported 9 . However, this study represents the first report of the effect of esculentin-2CHa(GA30) in attenuating and/or mitigating copper-induced neurotoxicity. The use of esculentin-2CHa(GA30) in this study is also predicated on previous reports of its beneficial effects in reducing oxidative stress in Drosophila melanogaster at concentrations tested in this study 13 . This study revealed that esculentin-2CHa(GA30) minimally reduced the lethality of copper and improved the eclosion rates in copper-treated and untreated D. melanogaster . Results of the present study also failed to show significant difference in the rate of mortality of D. melanogaster compared to previous studies in our laboratory 9 . These disparities could be attributed to the differences in the period of observation in this study (5 days) and in the previous study reported by Abolaji et al . 9 which observed treated flies for a longer period. These observations suggest that as the period of exposure of flies to copper increases, the rate of eclosion reduces, and by extension toxic effects of copper increases. Previous studies have established that at the cellular level, copper intoxication causes molecular damage via the generation of reactive oxygen species (ROS) and free radicals 21 . Copper toxicity has also been linked with the oxidation of biomolecules such as carbohydrates, nucleic acids, lipids, proteins, and other organic constituents of the living cell 22 . In this study, the exposure of flies to Cu 2+ led to elevated levels of H 2 O 2 level when compared to the control. This observation is consistent with the previous report which indicated that copper interacts with hydrogen peroxide (H 2 O 2 ) via a Fenton-like reaction resulting in the generation of free radicals such as hydroxyl radicals 9,21 . Consistent with previous antioxidative effects that have been reported for esculentin-2CHa(GA30) 13 , the peptide completely inhibited the effect of copper in elevating the levels of hydrogen peroxide in treated flies. These effects indicate that esculentin-2CHa(GA30) may exert its activities as a non-reducing substance to prevent free radical damage. Despite these antioxidative effects of the peptide, no significant effect of the peptide on copper ion-induced elevation of nitric oxide production was observed in this study. This may partly indicate that the antioxidative effect of the peptide may not involve the prevention of the accumulation of proinflammatory mediators such as nitric oxide. In addition, there is lack of clarity about the impact of excessive copper ion on nitric oxide generation in D. melanogaster . For instance, in a study which examined the anti-oxidative effects of curcumin in copper-treated flies, no significant increase in nitric oxide levels was observed in both copper-treated and untreated flies 9 . Therefore, further studies to actually delineate the effect of copper intoxication on nitric oxide production may be needed. Hydroxyl radicals exert inhibitory effects on enzymes and instigate lipid peroxidation reactions, leading to the disruption of cellular membranes and structure of organelles. Similarly, emulsions of unsaturated fatty acids following incubation with cupric chloride have been reported 23 . Elevated levels of cellular markers of lipid peroxide generation in liver homogenates of rats intoxicated with copper has also been reported 24 . These previous reports corroborate the increase in the level of TBARS in flies exposed to copper ions in this study. However, esculentin-2CHa(GA30) significantly inhibited the effect of copper on lipid peroxidation and even reduced basal lipid peroxidation that is not a consequent of copper intoxication. Effects observed for esculentin-2CHa(GA30) in this study is consistent with effects previously reported for many natural compounds including plant-derived material 25,26 and bioactive peptides 27,28,29 . The observed effect of esculentin-2CHa(GA30) on lipid peroxidation further highlights a potential role for the therapeutic benefits of the peptide in treating neurodegenerative disorders, as we have previously suggested 13 . The fact that many features characterizing many neurodegenerative disorders have been reported to include lipid peroxidation further supports this suggestion about the future therapeutic utility of esculentin-2CHa(GA30) 30 . The two most reactive products of lipid peroxidation (4-hydroxylnonenal, 4-HNE and trans-4-oxo-2-nonenal, 4-ONE) are involved in protein carbonylation, hence the assessment of the effect of esculentin-2CHa(GA30) on protein carbonylation in this study 31 . These metabolites diffuse from the membrane into the cytoplasm and nucleus where they covalently bind to cysteine, histidine, or lysine residues of proteins 32 . Carbonylation alters protein function, leading to deleterious intermolecular cross-links and aggregates that preclude their degradation by intracellular proteases 33 . Accumulation of carbonylated proteins has been implicated in the aetiology and/or progression of several chronic central nervous system (CNS) disorders including Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis 34 . This study reports effects of esculentin-2CHa(GA30) in inhibiting protein carbonylation induced by copper intoxication for the first time that, Therefore, it is possible that the peptide prevents in vivo aggregation lipid peroxidation metabolites that have implications for protein carbonylation. Enzymes such as catalase, superoxide dismutase and glutathione-S-transferase play significant roles in protecting cells from oxidative damage 35 . Actions of these enzymes are often supported by defensive mechanisms mediated by cellular constituents such as free amino acids, glutathione, and phenolic compounds 36 . It is against this background that the impact of copper intoxication on the activities of catalase and glutathione-s-transferase as well as levels of thiols in flies were examined. Consistent with other features of oxidative damage that have been highlighted in copper-intoxicated flies, reduced level of catalase and glutathione-s-transferase were observed in this study. Moreover, copper-intoxication was also associated with the depletion of non-protein and total thiols in D. melanogaster . However, these effects were inhibited in a dose-dependent manner in flies treated with esculentin-2CHa(GA30), further highlighting the potential therapeutic utility of the peptide in oxidative damage-related diseases. With respect to neurodegenerative disorders, Nandi et al. 37 has highlighted that the exploration of catalase activities could be a therapeutic target. Glutathione (GSH) chelates and detoxifies metals soon after they enter the cell 38 and could protect cells from deleterious effects such as those observed for copper-ions in this study. In fact, the role of glutathione in protecting against metal toxicity in rats 39 , mice 40 , cultured cells 41 , and Drosophila 9 have been reported. Therefore, the inhibition of the depletion of non-protein and total thiols by esculentin-2CHa(GA30) in this study further highlights the protective effect of the peptide against metal-induced toxicity and its beneficial effects in maintaining healthy antioxidant status. Studies involving Parkinson’s disease have reported the balance between the cholinergic and dopaminergic systems is required for normal functioning of the brain 42 . Acetylcholinesterase (AChE) is an important part of cholinergic system and is involved in the termination of neurotransmission via the breakdown of acetylcholine to acetate and choline 43 , and imbalances in acetylcholine metabolism has been linked with chronic conditions like Alzheimer’s disease and Parkinsonism 44 . These studies also highlight a significant role for AChE inhibitors in the management of neurodegenerative disorders 44 . In this study, esculentin-2CHa(GA30) significantly inhibited Cu 2+ -induced elevation of AChE activities in treated flies, indicating its beneficial actions in maintaining the normal functioning of the cholinergic system. In this study, the effect of esculentin-2CHa(GA30) on the activities of monoamine oxidase (MAO) was also assessed. This is against the background that the enzyme is involved in the removal of dopamine (also serotonin and norepinephrine) from the brain to maintain normal brain function 45 . Dopamine is released by the substantia nigra pars compacta of the brain and is essential for movement, memory, pleasurable reward, behavior and cognition, attention, inhibition of prolactin production, sleep, mood, and learning 46 . Consistent with these, monoamine oxidase inhibitors prevent neurotransmitter loss and preserve normal function of the brain 47 . The inhibition of monoamine oxidase activities by esculentin-2CHa(GA30) observed in this study therefore suggests that the peptide plays a significant role in maintaining the balance between the cholinergic and the dopaminergic systems. Consistent with the observed effects of esculentin-2CHa(GA30) on acetylcholinesterase and monoamine oxidase activities, improved motor activities observed in flies treated with the peptide is not surprising. Moreover, the observed improved negative geotaxis in treated flies may also be consequent on the antioxidative, free radical scavenging, and thiol-depletion preventing actions of the peptide. These activities of the peptide may have led to the restoration of impaired motor coordination and redox imbalance cause by copper-intoxication. Consistent with this assertion is the repopulation of brain neurons as well as the prevention of copper-induced cerebral atrophy observed in esculentin-2CHa(GA30) treated flies in this study. It is also possible that the peptide is able to sequester Cu 2+ in the microglia to prevent copper-induced damage to the brain. In conclusion, this study has established that esculentin-2CHa(GA30) may address copper-induced neurotoxicity from several dimensions, including the correction of cholinergic imbalance, preservation of brain neuronal distribution, restoration of healthy antioxidant status, and sequestration of metals to prevent metal-induced damage to the brain. These actions of esculentin-2CHa(GA30) open up a therapeutic window and motivate interests in further studies to develop therapeutic potential of the e use of the peptide in treating neurological disorders. MATERIALS AND METHODS Chemicals Copper sulfate was procured from AK Scientific, USA. Acetylthiocholine iodide, 1-chloro-2, 4-dinitrobenzene CDNB, and other chemicals used were purchased from Sigma USA and of high analytical grade. Peptide synthesis and purification The synthetic version of esculentin-2CHa(GA30) (> 95% pure) was purchased from a commercial vendor (Synpeptide Limited, Shanghai, China). The peptide was purified to homogeneity by reversed-phase high performance liquid chromatography (RP-HPLC) as previously described 13 . Briefly, the purification column (Vydac C18) was equilibrated with Solvent A containing trifluoroacetic acid (0.12%) in water and acetonitrile was used as the elution buffer. The gradient programme used involved increasing the concentration of acetonitrile in the elution buffer to 21% in 10 min, further to 56% in 15 min, and to 70% in another 15 min (total runtime = 40 min) at a flow rate of 1 ml/min. Absorbance was monitored at 254 nm and 280 nm. Chromeleon™ 7.3 CDS software was used for the analysis of the chromatogram obtained (Fig. 7 ). Drosophila melanogaster stock and culture Drosophila melanogaster wild-type (Harwich strain) flies were cultured and maintained in the Drosophila melanogaster Research Laboratory, Department of Biochemistry, College of Medicine, University of Ibadan, Oyo State, Nigeria. Flies were maintained on a measure of diet containing 52g of Cornmeal, 5g of brewer’s yeast, 7.9g of agar, and 0.7g of Nipagin. They were allowed to mate in vials monitored under a regulated temperature (22–24 \(℃\) ; 60–70% relative humidity) until the eggs metamorphosed into young adult fruit flies under a natural photoperiod of 12 hours light and 12 hours’ dark daily. Cu 2+ exposure and Esculentin-2CHa(GA30) treatment Based on previous studies in our laboratory, a dose of 1mM Cu 2+ derived from copper sulfate pentahydrate (CuSO 4 .5H 2 O) 9 , and two doses (5.0 and 7.5 µmol/kg diet) of esculentin-2CHa(GA30) 13 was used for this study. The mitigating action of esculentin-2CHa(GA30) on Cu 2+ -induced toxicity was assessed in flies (both genders, 1–3 days old) treated as summarized in Table 1 . Flies were exposed orally through their diet for five ( 5 ) days. Firstly, their survival rate was determined, and thereafter various biochemical parameters were assessed in this study Table 1 Experimental Design Group Name Treatment Group A Group B Group C Group D Group E Group F Diet only Diet + Esculenthin-2CHa(GA30) (5.0 µmol/kg diet) Diet + Esculenthin-2CHa(GA30) (7.5 µmol/kg diet) Diet + CuSO 4 (1mmol/kg diet) Diet + CuSO 4 (1mmol/kg diet) + Esculenthin-2CHa (5.0 µmol/kg diet) Diet + CuSO 4 (1mmol/kg diet) + Esculenthin-2CHa (7.5 µmol/kg diet) Survival and emergence rate of flies Flies (35 flies/vial, 5 replicates per group) were collected after anesthetisation, and placed into vials containing 4.9g of diet prepared as shown in Table 1 . The rate of survival was derived from data analysis of the daily records of the flies’ mortality. Also, the eclosion rate of D. melanogaster offspring after exposure of the parent flies to copper ions and esculentin-2CHA(GA30) was evaluated as previously described 9,14 . Preparation of samples for biochemical assays At the end of the treatment period treated flies (5 replicates per group) were anesthetised, collected, weighed, homogenized in 0.1 M phosphate buffer (pH 7.4) at a ratio of 1 mg:10µl of buffer. The homogenate was centrifuged at 4000 x g for 10 min at 4 o C in a Mikro 220R centrifuge (Tuttlingen, Germany). Subsequently, supernatants were retrieved and stored at -20 o C freezer until used for biochemical assays. All experiments were carried out in duplicates. Biochemical assays Determination of protein concentrations Total protein concentration was measured as previously described by Adesanoye et al. 13 . This method was a slight modification of the protocol described by Lowry et al. 48 . Briefly, fly homogenates (25 µl) were diluted ten times using distilled water. The diluted sample was subsequently mixed with Lowry’s reagent (400 µl). The mixture was then incubated at room temperature for 15 min. After the incubation period, the sample was diluted at a ratio of 1:5 with Folin Ciocalteau solution. The mixture was left at room temperature for 20 min. Absorbance readings were taken at 660nm. A standard curve made with graded concentrations of bovine serum albumin (BSA) was used for the interpolation of protein concentrations. Determination of nitric oxide (NO) levels The concentration of nitrite in the supernatant was measured as described by Yao et al 49 . Briefly, samples (200µl) were incubated with Griess reagent (200µl) in the dark at room temperature for 20 min. The absorbance was read at 550nm. Nitrite concentrations were extrapolated from a standard curve constructed using known concentration of sodium nitrite. Determination of hydrogen peroxide (H 2 O 2 ) levels Hydrogen peroxide generation was determined as described by Wolff 50 . Aliquots of homogenates (10µl) were mixed with 290µl of FOX reagent (10 ml of Xylenol Orange + 10 ml of sorbitol + 50 ml of AFS (Ammonium ferrous Sulfate) and 30 ml of distilled water. The reaction mixture was incubated at room temperature for 30 mins and the absorbance was read at 560nm. The concentration generated was extrapolated from the standard curve constructed using commercially available hydrogen peroxide. Measurement of lipid peroxidation (LPO) Lipid peroxidation was determined by the formation of thiobarbituric acid (TBA) reactive substances (TBARS) according to Varshney and Kale 51 method. The reaction medium contained 200 µl of trichloroacetic acid, 200 µl of thiobarbituric acid, and 100µl of fly homogenates. The mixture was incubated at 95 o C for 1h. After cooling to room temperature, the mixture was centrifuged at 8000 x g for 10 min. The absorbance of aliquots of supernatant (300µl) was read at 532nm. Measurement of protein carbonylation (PCO) The spectrophotometric method described by Augustyniak et al. 52 was used for the determination of absolute carbonyl levels. Homogenates (200µl) were mixed with equal volume of 2,4 dinitrophenylhydrazine (dissolved in 2.5M HCl). The mixture was vortexed and placed in the dark for 20 mins. Subsequently, 100 µl of 50% (w/v) Trichloroacetic acid was added to precipitate out the proteins. The mixture was incubated for 15 mins at -20 o C and centrifuged at 4 o C for 10 mins at 9000 rpm. The supernatant was discarded, and the pellet was washed twice in ice-cold 250 µl ethanol/ethyl acetate (1:1). The washed pellet was redissolved in 800µl of 6M guanidine hydrochloride. The absorbance was measured in the supernatant at 370nm, and carbonyl content was calculated. Determination of catalase (CAT) activity The method of Aebi 53 was used in determining catalase activity. The reaction mixture consisted of 50mM potassium phosphate buffer (pH 7.0), 300 mM H 2 O 2 and sample (1:50 dilution). The loss in absorbance of H 2 O 2 was monitored for 2 min at 240 nm and thereafter used to calculate catalase activity expressed as µmol of H 2 O 2 consumed per minute per milligram of protein. Determination of glutathione-s-transferase (GST) activity GST activity was determined as described by Habig et al. 54 with 1-chloro-2,4-dinitrobenzene (CDNB) used as the substrate. Homogenates (20µl) were added to a reagent mixture (270µl) made with 20ml of 0.25M potassium phosphate buffer (pH 7.0) containing EDTA at a final concentration of 2.5 mM, 10.5ml of distilled water and 500µl of 0.1 M GSH at 25°C), and 10 µl of 25 mM 2,4, dichlorobenzene (DCNB). Absorbance was measured at 340nm at 30s intervals for 2 min. Determination of non-protein thiol (NPSHs) level Levels of non-protein thiols (reduced gluthathione) were measured as described by Beutler et al. 55 . Briefly, 50µl of trichloroacetic acid was mixed with 50µl of sample. The mixture was incubated at 4 o C for 1 h prior to being centrifuged at 5000 x g for 10 minutes at 4 o C. Subsequently, 50µl of the supernatant was mixed with 250µl of 0.1M Phosphate buffer (pH 7.4) and 50µl of Ellman’s reagent (5,5′-dithiobis-(2-nitrobenzoic acid, DTNB). Absorbance was measured at 412 nm using the microplate reader. Behavioral /Neurotransmission Assay Determination of negative geotaxis This assay was used to investigate the effects of Cu 2+ and esculentin-2CHa(GA30) on the climbing rate of the flies. This was performed as previously described by Abolaji et al . 9 . Briefly, 30 flies per vial were treated and monitored for 5 days. After treatment, the flies were immobilized under mild ice. Anesthetized flies were placed (according to their groups) in labeled vertical glass columns (length 15cm, diameter 1.5cm). After recovery from the ice exposure, glass columns were gently tapped so that flies return to the bottom of the column. The number of flies that climbed up to the 6cm mark of the column within 6 seconds as well as those that were below this mark after the stipulated time were recorded. This procedure was repeated three times at 1min interval, and the climbing rate was calculated in percentage with respect to the total number of flies. Determination of acetylcholinesterase (AChE) activity Acetylcholinesterase activity was assayed as described by Ellman et al. 56 . Briefly, fly homogenates (10µl) were added to a reaction mixture containing 120µl of 0.1M potassium phosphate buffer (pH 7.4), 40µl of 10mM DTNB, 40µl of 8mM acetylthiocholine (the initiator) and 120µl of water. The reaction was monitored for 2 min (30 s intervals) at 412 nm. Data were calculated against reagent and sample blanks. The enzyme activity was estimated as µmol of acetylthiocholine hydrolyzed/minute/mg protein. Determination of monoamine oxidase (MAO) activity MAO activity was quantified using the method described by Tipton et al. 57 with slight modification. In a 2 ml Eppendorf tube, 80µl of 0.1M phosphate Buffer (pH 7.4), 260 µl of distilled H 2 O, 20 µl of 2.5mM benzylamine hydrochloride, and 40 µl of sample were mixed. The mixture was incubated for 30 mins at 25 o C. Afterward, 200 µl of perchloric acid was added to terminate the enzyme’s activity. The resulting solution was centrifuged at 1500 rpm for 10 mins. Absorbance was measured at 280nm. Histology D. melanogaster in control and each treatment group after five ( 5 ) days of treatment were fixed in 150µl of diluted Bouin solution (1 ml of Bouin + 10 ml of 0.1M PB) for 24 hours. Afterward, tflies were rinsed with 0.1M phosphate buffer (pH 7.4), until the yellow coloration of the Bouin solution was completely removed. Subsequently, rinsed flies were fixed in a solution of 10% Phosphate Buffer/Formalin. The flies were paraffinized and processed for hematoxylin and eosin (H&E) staining. An optic light microscope was used to view the brain. Statistical analysis Data were expressed as mean ± Standard Error of Mean (SEM). Treated and control groups were compared using one-way ANOVA. Significant differences between groups were detected using Turkeys Post-hoc test. Statistical significance was set at P < 0.05. All analyses were carried out using Graph Pad Prism Version 6.0 software for Windows. Declarations FUNDING No funding was received for this study DATA AVAILABILITY STATEMENT The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. CONFLICT OF INTERESTS 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 AUTHORSHIP OLU, OOO, and AOA contributed to the conception and design of the study, data interpretation and the preparation of manuscript. OLU, AO, AAF, OOO contributed to data collection and analysis. All authors approved the final manuscript. References Singh, R., Gautam, N., Mishra, A. and Gupta, R., 2011. Heavy metals and living systems: An overview. Indian journal of pharmacology, 43 (3), pp.246–253. Aguilar-Martinez, P., Grandchamp, B., Cunat, S., Cadet, E., Blanc, F., Nourrit, M., Lassoued, K., Schved, J.F. and Rochette, J., 2011. Iron overload in HFE C282Y heterozygotes at first genetic testing: a strategy for identifying rare HFE variants. haematologica , 96 (4), p.507. Kurutas, E.B., 2015. 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Pp 553–562 Sharma, A., Weber, D., Raupbach, J., Dakal, T.C., Fließbach, K., Ramirez, A., Grune, T. and Wüllner, U., 2020. Advanced glycation end products and protein carbonyl levels in plasma reveal sex-specific differences in Parkinson's and Alzheimer's disease. Redox biology, 34 , p.101546 Irato, P. and Santovito, G., 2021. Enzymatic and non-enzymatic molecules with antioxidant function. Antioxidants , 10 (4), p.579 Vaisi-Raygani, A., Rahimi, Z., Zahraie, M. and Pourmotabbed, M.N.A., 2007. Enzymatic and non-enzymatic antioxidant defense with Alzheimer disease. Acta Medica Iranica, pp.271–276. Nandi, A., Yan, L.J., Jana, C.K. and Das, N., 2019. Role of catalase in oxidative stress-and age-associated degenerative diseases. Oxidative medicine and cellular longevity , 2019 . Jozefczak, M., Remans, T., Vangronsveld, J. and Cuypers, A., 2012. Glutathione is a key player in metal-induced oxidative stress defenses. International journal of molecular sciences, 13 (3), pp.3145–3175. García, J.C., Remires, D., Leiva, A. and González, R., 2000. Depletion of brain glutathione potentiates the effect of 6-hydroxydopamine in a rat model of Parkinson’s disease. Journal of Molecular Neuroscience, 14 , pp.147–153. Sheweita, S.A., 1998. Heavy metal-induced changes in the Glutathione levels and Glutathione Reductase/Glutathione S-Transferase activities in the liver of male mice. International journal of toxicology, 17 (4), pp.383–392. Hultberg, B., Andersson, A. and Isaksson, A., 2001. Interaction of metals and thiols in cell damage and glutathione distribution: potentiation of mercury toxicity by dithiothreitol. Toxicology, 156 (2–3), pp.93–100. Bohnen, N.I. and Albin, R.L., 2011. The cholinergic system and Parkinson disease. Behavioural brain research, 221 (2), pp.564–573. Cox, M.A., Bassi, C., Saunders, M.E., Nechanitzky, R., Morgado-Palacin, I., Zheng, C. and Mak, T.W., 2020. Beyond neurotransmission: acetylcholine in immunity and inflammation. Journal of internal medicine, 287 (2), pp.120–133 Colovic, M.B., Krstic, D.Z., Lazarevic-Pasti, T.D., Bondzic, A.M. and Vasic, V.M., 2013. Acetylcholinesterase inhibitors: pharmacology and toxicology. Current neuropharmacology, 11 (3), pp.315–335. Meyer, J.H., Ginovart, N., Boovariwala, A., Sagrati, S., Hussey, D., Garcia, A., Young, T., Praschak-Rieder, N., Wilson, A.A. and Houle, S., 2006. Elevated monoamine oxidase a levels in the brain: an explanation for the monoamine imbalance of major depression. Archives of general psychiatry, 63 (11), pp.1209–1216. Olguín, J. H., Calderón Guzmán, D., Hernández García, E. and Barragán Mejía, G., 2016. The role of dopamine and its dysfunction as a consequence of oxidative stress. Oxidative medicine and cellular longevity , 2016 . Jones, D.N. and Raghanti, M.A., 2021. The role of monoamine oxidase enzymes in the pathophysiology of neurological disorders. Journal of Chemical Neuroanatomy , 114 , p.101957 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J biol Chem, 193 (1), pp.265–275. Yao, D., Vlessidis, A.G. and Evmiridis, N.P., 2004. Determination of nitric oxide in biological samples. Microchimica Acta, 147 , pp.1–20 Wolff, S.P., 1994. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. In Methods in enzymology (Vol. 233, pp. 182–189). Academic Press Varshney, R. and Kale, R.K., 1990. Effects of calmodulin antagonists on radiation-induced lipid peroxidation in microsomes. International journal of radiation biology, 58 (5), pp.733–743. Augustyniak, E., Adam, A., Wojdyla, K., Rogowska-Wrzesinska, A., Willetts, R., Korkmaz, A., Atalay, M., Weber, D., Grune, T., Borsa, C. and Gradinaru, D., 2015. Validation of protein carbonyl measurement: a multi-centre study. Redox biology, 4 , pp.149–157 Aebi, H., 1984. [13] Catalase in vitro. In Methods in enzymology (Vol. 105, pp. 121–126). Academic press. Habig, W.H., Pabst, M.J. and Jakoby, W.B., 1974. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. Journal of biological Chemistry, 249 (22), pp.7130–7139 Beutler, E., Duron, O. and Kelly, B.M., 1963. Improved method for the determination of blood glutathione. The Journal of Laboratory and Clinical Medicine, 61 , pp.882–888 Ellman, G.L., Courtney, K.D., Andres Jr, V. and Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical pharmacology, 7 (2), pp.88–95 Tipton, K.F., Davey, G. and Motherway, M., 2000. Monoamine oxidase assays. Current protocols in pharmacology, 9 (1), pp.3–6 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4368804","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":301942090,"identity":"9b4b676d-f9d1-4702-b4d5-4e86ff21f928","order_by":0,"name":"Onyedika L. 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It facilitates several physiological processes including antioxidant defense, collagen synthesis, maintenance of blood vessel integrity, oxygen metabolism, skin pigmentation, synthesis of neurotransmitters, and the regulation of iron homeostasis\u003csup\u003e2\u003c/sup\u003e. However, emerging evidence indicates that the redox activity that makes copper physiologically essential is deleterious at excessive cellular levels of copper ions\u003csup\u003e3,4\u003c/sup\u003e. Aberration in copper homeostasis may result from aging, environmental influences, or genetic mutation and this has been implicated in a variety of abnormal physiological conditions like cancer, inflammation, and neurodegeneration\u003csup\u003e5,6,7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, the pathology of Alzheimer\u0026rsquo;s disease (AD) has been traced to the imbalances of intracellular copper homeostasis and it has been shown that exposure to high copper concentration could lead to increased Amyloid Precursor Protein (APP) expression, amyloid beta (Aβ) aggregation, and hyperphosphorylation of tau\u003csup\u003e8\u003c/sup\u003e. Similarly, previous studies revealed that excessive copper intake affects cognition and plays a role in the pathogenesis of AD, Parkinson Disease, and amyotrophic lateral sclerosis\u003csup\u003e9\u003c/sup\u003e. Cruces-Sande \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e assessed \u003cem\u003ein vivo\u003c/em\u003e effects of copper in brain oxidative damage and its ability to increase dopaminergic degeneration induced by 6-hydroxydopamine. The study reported that chronic copper administration caused the accumulation of 6-hydroxydopamine in different areas of the brain (cortex, striatum, nigra). The accumulation of 6-hydroxydopamine was also observed to be associated with increased levels of Thiobarbituric Reactive Substance (TBARS), decreased levels of protein free-thiol in the cortex, reduced catalase activity and increased in glutathione peroxidase activity\u003csup\u003e10\u003c/sup\u003e. This indicates that copper potentiates the action of other factors involved in the pathogenesis of neurodegenerative diseases through oxidative stress. The quest to preserve copper homeostasis opened up new therapeutic avenues in the definition of the novel disease-modifying bioactive peptide, esculentin-2CHa(GA30) which has a dearth of information on its neuroprotective potential.\u003c/p\u003e \u003cp\u003eEsculentin-2CHa (GFSSIFRGVAKFASKGLGKDLAKLGVDLVA) is the truncated version of esculentin-2CHa (which has 37 amino acid residues) originally isolated from the skin secretion of the Chiricahua leopard frog, \u003cem\u003eLithobates chiricahuensis\u003c/em\u003e\u003csup\u003e11\u003c/sup\u003e. The frog skin host-defense peptide, esculentin-2CHa has been shown to display multifunctional effects including antimicrobial, antitumor, and immunomodulatory properties\u003csup\u003e11\u003c/sup\u003e. Esculentin-2CHa(GA30) is the truncated version of the original peptide, lacking the last seven amino acid residues\u003csup\u003e12\u003c/sup\u003e. Antidiabetic\u003csup\u003e12\u003c/sup\u003e and antioxidative effects\u003csup\u003e13\u003c/sup\u003e of esculentin-2CHa(GA30) have been reported. These previously reported biological effects motivated the selection of esculentin-2CHa(GA30) and the hypothesis that esculentin-2CHa(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) will elicit neuroprotective effects against copper toxicity.\u003c/p\u003e \u003cp\u003eAlthough most studies involving metal toxicity and neurodegeneration use vertebrate models (humans/ rodents). However, invertebrate models are now been considered as alternative useful and cheaper models. In this study, \u003cem\u003eDrosophila melanogaster\u003c/em\u003e was selected as a model organism due to its extensive metabolic and genetic similarity to humans\u003csup\u003e14\u003c/sup\u003e. It has been reported that \u003cem\u003eD. melanogaster\u003c/em\u003e is about 60% genetically similar to humans and has several similar biological mechanisms\u003csup\u003e9,14\u003c/sup\u003e. In this study, we investigated neuroprotective effects as well as the ability of esculentin-2CHa(GA30) to protect D. melanogaster against oxidative damage and neuro-behavioural deficit associated with copper-induced toxicity.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffect of esculentin-2CHa(GA30) on survival and eclosion rates in Drosophila melanogaster\u003c/h2\u003e \u003cp\u003eThe rate at which control flies as well as flies treated with Cu\u003csup\u003e2+\u003c/sup\u003e in the absence or presence of esculentin-2CHa(GA30) die over a 5-day period is presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Data presented in these Figures indicate a similar death rate in both groups. However, there are differences in the number of surviving flies across the groups on Day 5. Specifically, 26.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 flies in the control group survived up to Day 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This is similar to the average number of surviving flies in groups treated with the peptide at 5.0\u0026micro;M/kg diet (25.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 flies) and 7.5\u0026micro;M/kg diet (27.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 flies). The treatment of flies with Cu\u003csup\u003e2+\u003c/sup\u003e reduced the number of surviving flies by 14.3% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This reduction was prevented by treated of flies with the peptide at both 5.0\u0026micro;M/kg diet (25.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3 flies) and 7.5\u0026micro;M/kg diet (24.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 flies).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe impact of the exposure of flies to Cu\u003csup\u003e2+\u003c/sup\u003e on eclosion rates was more pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The rate of emergence of new flies in Cu\u003csup\u003e2+\u003c/sup\u003e treated groups reduced by 34.8% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the control group. Eclosion rate in the group treated with the peptide at 5.0\u0026micro;M/kg diet was similar to the control group. At the peptide concentration of 7.5\u0026micro;M/kg diet, eclosion rate increased by 1.6-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the absence of Cu\u003csup\u003e2+\u003c/sup\u003e and by 1.1-fold (ns) in the presence of Cu\u003csup\u003e2+\u003c/sup\u003e compared to control flies. The effect observed in the group treated with peptide (7.5\u0026micro;M/kg diet) and Cu\u003csup\u003e2+\u003c/sup\u003e represent a 1.7-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) increase compared to flies treated with Cu\u003csup\u003e2+\u003c/sup\u003e alone.\u003c/p\u003e \u003cp\u003eWith respect to the number of emerging flies on Day 12, a similar number of emerging flies was observed in the control and the Cu\u003csup\u003e2+\u003c/sup\u003e groups. The number of emerging flies in the group treated with 5.0\u0026micro;M/kg diet alone is also similar to the number observed in the control group and represent a 12.3% (ns) increase compared to the number of emerging flies observed in the group treated with Cu\u003csup\u003e2+\u003c/sup\u003e at the same peptide concentration. Compared to all other groups, the number of emerging flies was significantly higher in the group treated with the peptide alone at the dose of 7.5\u0026micro;M/kg diet (1.5-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The effect of Cu\u003csup\u003e2+\u003c/sup\u003e on the number of emerging flies was also completely inhibited at this peptide concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of esculentin-2CHa(GA30) on H\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eO\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eand NO\u003c/b\u003e \u003cb\u003elevels in\u003c/b\u003e \u003cb\u003eCu\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e-treated Drosophila melanogaster\u003c/b\u003e\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NO levels were assessed as markers of oxidative stress in this study. Results obtained indicate that treatment of flies with Cu\u003csup\u003e2+\u003c/sup\u003e led to 16.2% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) compared to control flies. Unlike Cu\u003csup\u003e2+\u003c/sup\u003e, the treatment of flies with esculentin-2CHa(GA30) alone at all concentrations tested did not produce significant increase in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels. However, the elevation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels produced by Cu\u003csup\u003e2+\u003c/sup\u003e was reduced by 15.8% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 15.1% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in flies treated with 5.0 and 7.5 \u0026micro;M/kg diet of esculentin-2CHa(GA30) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). NO levels in control flies and flies treated with esculentin-2CHa(GA30) were similar. However, treated with Cu\u003csup\u003e2+\u003c/sup\u003e increased NO levels by 36.4% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The combination of Cu\u003csup\u003e2+\u003c/sup\u003e with esculentin-2CHa(GA30) did not reduce the elevation in NO levels produced by Cu\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEffects of esculentin-2CHa(GA30) on lipid peroxidation and protein carbonylation in Cu\u003csup\u003e2+\u003c/sup\u003e-treated Drosophila melanogaster\u003c/h2\u003e \u003cp\u003eThe concentration of thiobarbituric reactive substances (TBARS) in control flies was observed as 36.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6 nmol/mg protein. The concentration of TBARS was increased by 32.8% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in flies exposed to Cu\u003csup\u003e2+\u003c/sup\u003e only (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The treatment of flies with esculentin-2CHa(GA30) at both dosages used in this study significantly reduced basal and Cu\u003csup\u003e2+\u003c/sup\u003e-induced accumulation of TBARS. Specifically, TBARS in flies treated with the peptide alone at 5.0\u0026micro;M/kg diet was 17.3% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) lower compared to control flies. The peptide at 5.0\u0026micro;M/kg diet reduced Cu\u003csup\u003e2+\u003c/sup\u003e-induced elevation of TBARS by 37.2% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A more significant reduction in basal (40.8%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and Cu\u003csup\u003e2+\u003c/sup\u003e-induced (55.1%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) TBARS levels was observed in flies treated with esculentin-2CHa(GA30) at 7.5\u0026micro;M/kg diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similarly, protein carbonyl levels in Cu\u003csup\u003e2+\u003c/sup\u003e-treated flies in the absence of the peptide increased by 1.5-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared with control flies. However, the treatment of flies with esculentin-2CHa(GA30) at 5.0\u0026micro;M/kg diet reduced basal (20.7%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and Cu\u003csup\u003e2+\u003c/sup\u003e-induced (47.0%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) levels of protein carbonyls. Similarly, reduced basal (33.6%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and Cu\u003csup\u003e2+\u003c/sup\u003e-induced (63.8%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) levels of protein carbonyls were observed in flies treated with the peptide at 7.5\u0026micro;M/kg diet (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Esculentin-2CHa(GA30) on activities of catalase and glutathione-S-transferase in Cu\u003csup\u003e2+\u003c/sup\u003e-treated Drosophila melanogaster\u003c/h2\u003e \u003cp\u003eBasal catalase activities in control flies were estimated as 90.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 nmol/mg protein. This was significantly reduced by the exposure of flies to Cu2+ (74.0%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In the presence of peptide alone at 5.0\u0026micro;M/kg diet, a reduction of 28.6% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in catalase activities was also observed. However, the combination of esculentin-2CHa(GA30) (5.0\u0026micro;M/kg diet) with Cu\u003csup\u003e2+\u003c/sup\u003e resulted in the inhibition of Cu\u003csup\u003e2+\u003c/sup\u003e-induced reduction in catalase activity (55.7%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). At peptide concentration of 7.5\u0026micro;M/kg diet alone, an increase of 1.7-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed compared with control flies. When the peptide at 7.5\u0026micro;M/kg diet was combined with Cu2+, the deleterious effects of Cu2\u0026thinsp;+\u0026thinsp;on catalase activities was completely reversed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, glutathione-s-transferase activities in control flies were estimated as 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 nmol/kg protein. These activities were reduced by 52.7% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in flies exposed to Cu2+. Treatment with esculentin-2CHa(GA30) alone increased glutathione-s-transferase activities by 1.6-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 2.1-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at peptide concentrations of 5.0 and 7.5 \u0026micro;M/kg diet respectively. In the presence of Cu2+, the peptide inhibited the deleterious effects of Cu2\u0026thinsp;+\u0026thinsp;by 67.2% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 86.6% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEffects of esculentin-2CHa(GA30) on levels of non-protein thiols and total thiols in Cu\u003csup\u003e2+\u003c/sup\u003e-treated Drosophila melanogaster\u003c/h2\u003e \u003cp\u003eThe treatment of flies with esculentin-2CHa(GA30) at 5.0\u0026micro;M/kg diet did not affect the level of non-protein thiols when compared with control flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). A similar result was obtained at the peptide concentration of 7.5\u0026micro;M/kg diet. However, the level of non-protein thiols in flies treated with Cu\u003csup\u003e2+\u003c/sup\u003e was reduced by 27.7% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Esculentin-2CHa(GA30) at 5.0\u0026micro;M/kg diet increased the level of non-protein thiols by 13.6% (ns) compared to Cu\u003csup\u003e2+\u003c/sup\u003e-treated flies and at a peptide concentration of 7.5\u0026micro;M/kg diet, the deleterious effect of Cu\u003csup\u003e2+\u003c/sup\u003e was completely inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The treatment of flies with esculentin-2CHa(GA30) at 5.0 and 7.5 \u0026micro;M/kg diet increased total thiols levels by 26.0% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 23.3% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) when compared with control flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The treatment of flies with Cu2\u0026thinsp;+\u0026thinsp;also reduced total thiol concentration by 21.2% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The peptide failed to completely inhibit the effect of Cu\u003csup\u003e2+\u003c/sup\u003e, even though total thiol levels increased by 1.2-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 1.3-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in flies treated with the peptide at 5.0 and 7.5 \u0026micro;M/kg diet respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEffect of esculentin-2CHa(GA30) on behavioural and neurotransmission markers in Drosophila melanogaster\u003c/h2\u003e \u003cp\u003eNegative geotaxis was observed in 74.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4% of control flies. The treatment of flies with esculentin-2CHa(GA30) did not significantly change the number of flies with the ability for negative geotaxis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, following the exposure of flies to Cu\u003csup\u003e2+\u003c/sup\u003e, only 52.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.1% of flies had the ability for negative geotaxis. Treatment of flies exposed to Cu\u003csup\u003e2+\u003c/sup\u003e with esculentin-2CHa(GA30) at 5.0\u0026micro;M/kg diet completely prevented the effect of Cu\u003csup\u003e2+\u003c/sup\u003e on negative geotaxis. At peptide concentration of 7.5\u0026micro;M/kg diet, the number of flies with the ability for negative geotaxis increased by 21.4% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to control flies and by 72.1% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to untreated flies exposed to Cu\u003csup\u003e2+\u003c/sup\u003e alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMonoamine oxidase activities in control flies was estimated as 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 nmol/mg protein. These activities were reduced by 13.6% (ns) and 41.5% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to control flies. The exposure of flies to Cu\u003csup\u003e2+\u003c/sup\u003e, increased monoamine oxidase activities by 1.4-fold (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, the treatment of flies exposed to Cu\u003csup\u003e2+\u003c/sup\u003e with esculentin-2CHa(GA30) at 5.0 and 7.5 \u0026micro;M/kg diet completely inhibited the effect of Cu\u003csup\u003e2+\u003c/sup\u003e and reduced monoamine oxidase by 21.7% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 39.7% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) respectively when compared to control flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Similar effects were observed for acetylcholine esterase activities. Specifically, similar acetylcholine esterase activities were observed in control flies and flies treated with esculentin-2CHa(GA30) at 5.0 and 7.5 \u0026micro;M/kg diet alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Increased enzyme activities (1.9-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in flies exposed to Cu\u003csup\u003e2+\u003c/sup\u003e. The treatment of Cu\u003csup\u003e2+\u003c/sup\u003e-exposed flies with esculentin-2CHa(GA30) reduced the effect of Cu\u003csup\u003e2+\u003c/sup\u003e by 40.1% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and 55.9% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) at 5.0 and 7.5 \u0026micro;M/kg diet peptide concentrations respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCopper-chelating effects of esculentin-2CHa(GA30) in the brain of Drosophila melanogaster\u003c/h2\u003e \u003cp\u003eThe brain photomicrograph taken across the treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) indicated an even distribution of the neurons around the white matter of control flies and flies treated with esculentin-2CHa(GA30) at all concentrations tested. However, there is atrophy of neurons and white matter in the brain of flies exposed to Cu\u003csup\u003e2+\u003c/sup\u003e. A moderate and significant repopulation of neurons in the grey matter of the lobular of flies co-treated with the peptide and Cu\u003csup\u003e2+\u003c/sup\u003e 5.0\u0026micro;M and 7.5 \u0026micro;M respectively was observed was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eCopper is an essential metabolic trace element in all organisms, and it serves as cofactors for many enzymatic reactions\u003csup\u003e15,16\u003c/sup\u003e. However, deleterious effects of excessive copper levels have been reported\u003csup\u003e9\u003c/sup\u003e. The ease of transition between the mono- and the di-ionic states of copper, resulting in the generation of free radicals and oxidative stress, has been implicated in the toxic effects of copper\u003csup\u003e15,17\u003c/sup\u003e. Moreso, imbalance in copper homeostasis has been associated with neurodegeneration due to its ability to bind to hyperphosphorylated tau proteins in neurons\u003csup\u003e18\u003c/sup\u003e. This prescribes a role for imbalance in copper homeostasis in the pathogenesis of diseases such as Alzheimer\u0026rsquo;s and Parkinson\u0026rsquo;s diseases. The fact that \u003cem\u003eDrosophila\u003c/em\u003e melanogaster possesses unique characteristics such as ease of handling, short life span, genetic tractability, complex behaviors, and well-known simple neuroanatomy made it a good model for neurological studies, such as the one reported in this article\u003csup\u003e19,20\u003c/sup\u003e. The neurotoxic effect of copper in \u003cem\u003eD. melanogaster\u003c/em\u003e has been reported\u003csup\u003e9\u003c/sup\u003e. However, this study represents the first report of the effect of esculentin-2CHa(GA30) in attenuating and/or mitigating copper-induced neurotoxicity. The use of esculentin-2CHa(GA30) in this study is also predicated on previous reports of its beneficial effects in reducing oxidative stress in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e at concentrations tested in this study\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study revealed that esculentin-2CHa(GA30) minimally reduced the lethality of copper and improved the eclosion rates in copper-treated and untreated \u003cem\u003eD. melanogaster\u003c/em\u003e. Results of the present study also failed to show significant difference in the rate of mortality of \u003cem\u003eD. melanogaster\u003c/em\u003e compared to previous studies in our laboratory\u003csup\u003e9\u003c/sup\u003e. These disparities could be attributed to the differences in the period of observation in this study (5 days) and in the previous study reported by Abolaji \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e9\u003c/sup\u003e which observed treated flies for a longer period. These observations suggest that as the period of exposure of flies to copper increases, the rate of eclosion reduces, and by extension toxic effects of copper increases.\u003c/p\u003e \u003cp\u003ePrevious studies have established that at the cellular level, copper intoxication causes molecular damage via the generation of reactive oxygen species (ROS) and free radicals\u003csup\u003e21\u003c/sup\u003e. Copper toxicity has also been linked with the oxidation of biomolecules such as carbohydrates, nucleic acids, lipids, proteins, and other organic constituents of the living cell\u003csup\u003e22\u003c/sup\u003e. In this study, the exposure of flies to Cu\u003csup\u003e2+\u003c/sup\u003e led to elevated levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e level when compared to the control. This observation is consistent with the previous report which indicated that copper interacts with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) via a Fenton-like reaction resulting in the generation of free radicals such as hydroxyl radicals\u003csup\u003e9,21\u003c/sup\u003e. Consistent with previous antioxidative effects that have been reported for esculentin-2CHa(GA30)\u003csup\u003e13\u003c/sup\u003e, the peptide completely inhibited the effect of copper in elevating the levels of hydrogen peroxide in treated flies. These effects indicate that esculentin-2CHa(GA30) may exert its activities as a non-reducing substance to prevent free radical damage.\u003c/p\u003e \u003cp\u003eDespite these antioxidative effects of the peptide, no significant effect of the peptide on copper ion-induced elevation of nitric oxide production was observed in this study. This may partly indicate that the antioxidative effect of the peptide may not involve the prevention of the accumulation of proinflammatory mediators such as nitric oxide. In addition, there is lack of clarity about the impact of excessive copper ion on nitric oxide generation in \u003cem\u003eD. melanogaster\u003c/em\u003e. For instance, in a study which examined the anti-oxidative effects of curcumin in copper-treated flies, no significant increase in nitric oxide levels was observed in both copper-treated and untreated flies\u003csup\u003e9\u003c/sup\u003e. Therefore, further studies to actually delineate the effect of copper intoxication on nitric oxide production may be needed.\u003c/p\u003e \u003cp\u003eHydroxyl radicals exert inhibitory effects on enzymes and instigate lipid peroxidation reactions, leading to the disruption of cellular membranes and structure of organelles. Similarly, emulsions of unsaturated fatty acids following incubation with cupric chloride have been reported\u003csup\u003e23\u003c/sup\u003e. Elevated levels of cellular markers of lipid peroxide generation in liver homogenates of rats intoxicated with copper has also been reported\u003csup\u003e24\u003c/sup\u003e. These previous reports corroborate the increase in the level of TBARS in flies exposed to copper ions in this study. However, esculentin-2CHa(GA30) significantly inhibited the effect of copper on lipid peroxidation and even reduced basal lipid peroxidation that is not a consequent of copper intoxication. Effects observed for esculentin-2CHa(GA30) in this study is consistent with effects previously reported for many natural compounds including plant-derived material\u003csup\u003e25,26\u003c/sup\u003e and bioactive peptides\u003csup\u003e27,28,29\u003c/sup\u003e. The observed effect of esculentin-2CHa(GA30) on lipid peroxidation further highlights a potential role for the therapeutic benefits of the peptide in treating neurodegenerative disorders, as we have previously suggested\u003csup\u003e13\u003c/sup\u003e. The fact that many features characterizing many neurodegenerative disorders have been reported to include lipid peroxidation further supports this suggestion about the future therapeutic utility of esculentin-2CHa(GA30)\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe two most reactive products of lipid peroxidation (4-hydroxylnonenal, 4-HNE and trans-4-oxo-2-nonenal, 4-ONE) are involved in protein carbonylation, hence the assessment of the effect of esculentin-2CHa(GA30) on protein carbonylation in this study\u003csup\u003e31\u003c/sup\u003e. These metabolites diffuse from the membrane into the cytoplasm and nucleus where they covalently bind to cysteine, histidine, or lysine residues of proteins\u003csup\u003e32\u003c/sup\u003e. Carbonylation alters protein function, leading to deleterious intermolecular cross-links and aggregates that preclude their degradation by intracellular proteases\u003csup\u003e33\u003c/sup\u003e. Accumulation of carbonylated proteins has been implicated in the aetiology and/or progression of several chronic central nervous system (CNS) disorders including Alzheimer\u0026rsquo;s disease, Parkinson\u0026rsquo;s disease, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis\u003csup\u003e34\u003c/sup\u003e. This study reports effects of esculentin-2CHa(GA30) in inhibiting protein carbonylation induced by copper intoxication for the first time that, Therefore, it is possible that the peptide prevents \u003cem\u003ein vivo\u003c/em\u003e aggregation lipid peroxidation metabolites that have implications for protein carbonylation.\u003c/p\u003e \u003cp\u003eEnzymes such as catalase, superoxide dismutase and glutathione-S-transferase play significant roles in protecting cells from oxidative damage\u003csup\u003e35\u003c/sup\u003e. Actions of these enzymes are often supported by defensive mechanisms mediated by cellular constituents such as free amino acids, glutathione, and phenolic compounds\u003csup\u003e36\u003c/sup\u003e. It is against this background that the impact of copper intoxication on the activities of catalase and glutathione-s-transferase as well as levels of thiols in flies were examined. Consistent with other features of oxidative damage that have been highlighted in copper-intoxicated flies, reduced level of catalase and glutathione-s-transferase were observed in this study. Moreover, copper-intoxication was also associated with the depletion of non-protein and total thiols in \u003cem\u003eD. melanogaster\u003c/em\u003e. However, these effects were inhibited in a dose-dependent manner in flies treated with esculentin-2CHa(GA30), further highlighting the potential therapeutic utility of the peptide in oxidative damage-related diseases. With respect to neurodegenerative disorders, Nandi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e37\u003c/sup\u003e has highlighted that the exploration of catalase activities could be a therapeutic target.\u003c/p\u003e \u003cp\u003eGlutathione (GSH) chelates and detoxifies metals soon after they enter the cell\u003csup\u003e38\u003c/sup\u003e and could protect cells from deleterious effects such as those observed for copper-ions in this study. In fact, the role of glutathione in protecting against metal toxicity in rats\u003csup\u003e39\u003c/sup\u003e, mice\u003csup\u003e40\u003c/sup\u003e, cultured cells\u003csup\u003e41\u003c/sup\u003e, and \u003cem\u003eDrosophila\u003c/em\u003e\u003csup\u003e9\u003c/sup\u003e have been reported. Therefore, the inhibition of the depletion of non-protein and total thiols by esculentin-2CHa(GA30) in this study further highlights the protective effect of the peptide against metal-induced toxicity and its beneficial effects in maintaining healthy antioxidant status.\u003c/p\u003e \u003cp\u003eStudies involving Parkinson\u0026rsquo;s disease have reported the balance between the cholinergic and dopaminergic systems is required for normal functioning of the brain\u003csup\u003e42\u003c/sup\u003e. Acetylcholinesterase (AChE) is an important part of cholinergic system and is involved in the termination of neurotransmission via the breakdown of acetylcholine to acetate and choline\u003csup\u003e43\u003c/sup\u003e, and imbalances in acetylcholine metabolism has been linked with chronic conditions like Alzheimer\u0026rsquo;s disease and Parkinsonism\u003csup\u003e44\u003c/sup\u003e. These studies also highlight a significant role for AChE inhibitors in the management of neurodegenerative disorders\u003csup\u003e44\u003c/sup\u003e. In this study, esculentin-2CHa(GA30) significantly inhibited Cu\u003csup\u003e2+\u003c/sup\u003e-induced elevation of AChE activities in treated flies, indicating its beneficial actions in maintaining the normal functioning of the cholinergic system.\u003c/p\u003e \u003cp\u003eIn this study, the effect of esculentin-2CHa(GA30) on the activities of monoamine oxidase (MAO) was also assessed. This is against the background that the enzyme is involved in the removal of dopamine (also serotonin and norepinephrine) from the brain to maintain normal brain function\u003csup\u003e45\u003c/sup\u003e. Dopamine is released by the substantia nigra pars compacta of the brain and is essential for movement, memory, pleasurable reward, behavior and cognition, attention, inhibition of prolactin production, sleep, mood, and learning\u003csup\u003e46\u003c/sup\u003e. Consistent with these, monoamine oxidase inhibitors prevent neurotransmitter loss and preserve normal function of the brain\u003csup\u003e47\u003c/sup\u003e. The inhibition of monoamine oxidase activities by esculentin-2CHa(GA30) observed in this study therefore suggests that the peptide plays a significant role in maintaining the balance between the cholinergic and the dopaminergic systems.\u003c/p\u003e \u003cp\u003eConsistent with the observed effects of esculentin-2CHa(GA30) on acetylcholinesterase and monoamine oxidase activities, improved motor activities observed in flies treated with the peptide is not surprising. Moreover, the observed improved negative geotaxis in treated flies may also be consequent on the antioxidative, free radical scavenging, and thiol-depletion preventing actions of the peptide. These activities of the peptide may have led to the restoration of impaired motor coordination and redox imbalance cause by copper-intoxication. Consistent with this assertion is the repopulation of brain neurons as well as the prevention of copper-induced cerebral atrophy observed in esculentin-2CHa(GA30) treated flies in this study. It is also possible that the peptide is able to sequester Cu\u003csup\u003e2+\u003c/sup\u003e in the microglia to prevent copper-induced damage to the brain.\u003c/p\u003e \u003cp\u003eIn conclusion, this study has established that esculentin-2CHa(GA30) may address copper-induced neurotoxicity from several dimensions, including the correction of cholinergic imbalance, preservation of brain neuronal distribution, restoration of healthy antioxidant status, and sequestration of metals to prevent metal-induced damage to the brain. These actions of esculentin-2CHa(GA30) open up a therapeutic window and motivate interests in further studies to develop therapeutic potential of the e use of the peptide in treating neurological disorders.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eCopper sulfate was procured from AK Scientific, USA. Acetylthiocholine iodide, 1-chloro-2, 4-dinitrobenzene CDNB, and other chemicals used were purchased from Sigma USA and of high analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePeptide synthesis and purification\u003c/h2\u003e \u003cp\u003eThe synthetic version of esculentin-2CHa(GA30) (\u0026gt;\u0026thinsp;95% pure) was purchased from a commercial vendor (Synpeptide Limited, Shanghai, China). The peptide was purified to homogeneity by reversed-phase high performance liquid chromatography (RP-HPLC) as previously described\u003csup\u003e13\u003c/sup\u003e. Briefly, the purification column (Vydac C18) was equilibrated with Solvent A containing trifluoroacetic acid (0.12%) in water and acetonitrile was used as the elution buffer. The gradient programme used involved increasing the concentration of acetonitrile in the elution buffer to 21% in 10 min, further to 56% in 15 min, and to 70% in another 15 min (total runtime\u0026thinsp;=\u0026thinsp;40 min) at a flow rate of 1 ml/min. Absorbance was monitored at 254 nm and 280 nm. Chromeleon\u0026trade; 7.3 CDS software was used for the analysis of the chromatogram obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDrosophila melanogaster stock and culture\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDrosophila melanogaster\u003c/em\u003e wild-type (Harwich strain) flies were cultured and maintained in the \u003cem\u003eDrosophila melanogaster\u003c/em\u003e Research Laboratory, Department of Biochemistry, College of Medicine, University of Ibadan, Oyo State, Nigeria. Flies were maintained on a measure of diet containing 52g of Cornmeal, 5g of brewer\u0026rsquo;s yeast, 7.9g of agar, and 0.7g of Nipagin. They were allowed to mate in vials monitored under a regulated temperature (22\u0026ndash;24 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e; 60\u0026ndash;70% relative humidity) until the eggs metamorphosed into young adult fruit flies under a natural photoperiod of 12 hours light and 12 hours\u0026rsquo; dark daily.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCu\u003csup\u003e2+\u003c/sup\u003e exposure and Esculentin-2CHa(GA30) treatment\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBased on previous studies in our laboratory, a dose of 1mM Cu\u003csup\u003e2+\u003c/sup\u003e derived from copper sulfate pentahydrate (CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO)\u003csup\u003e9\u003c/sup\u003e, and two doses (5.0 and 7.5 \u0026micro;mol/kg diet) of esculentin-2CHa(GA30)\u003csup\u003e13\u003c/sup\u003e was used for this study. The mitigating action of esculentin-2CHa(GA30) on Cu\u003csup\u003e2+\u003c/sup\u003e-induced toxicity was assessed in flies (both genders, 1\u0026ndash;3 days old) treated as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Flies were exposed orally through their diet for five (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) days. Firstly, their survival rate was determined, and thereafter various biochemical parameters were assessed in this study\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental Design\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup A\u003c/p\u003e \u003cp\u003eGroup B\u003c/p\u003e \u003cp\u003eGroup C\u003c/p\u003e \u003cp\u003eGroup D\u003c/p\u003e \u003cp\u003eGroup E\u003c/p\u003e \u003cp\u003eGroup F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiet only\u003c/p\u003e \u003cp\u003eDiet\u0026thinsp;+\u0026thinsp;Esculenthin-2CHa(GA30) (5.0 \u0026micro;mol/kg diet)\u003c/p\u003e \u003cp\u003eDiet\u0026thinsp;+\u0026thinsp;Esculenthin-2CHa(GA30) (7.5 \u0026micro;mol/kg diet)\u003c/p\u003e \u003cp\u003eDiet\u0026thinsp;+\u0026thinsp;CuSO\u003csub\u003e4\u003c/sub\u003e (1mmol/kg diet)\u003c/p\u003e \u003cp\u003eDiet\u0026thinsp;+\u0026thinsp;CuSO\u003csub\u003e4\u003c/sub\u003e (1mmol/kg diet)\u0026thinsp;+\u0026thinsp;Esculenthin-2CHa (5.0 \u0026micro;mol/kg diet)\u003c/p\u003e \u003cp\u003eDiet\u0026thinsp;+\u0026thinsp;CuSO\u003csub\u003e4\u003c/sub\u003e (1mmol/kg diet)\u0026thinsp;+\u0026thinsp;Esculenthin-2CHa (7.5 \u0026micro;mol/kg diet)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSurvival and emergence rate of flies\u003c/h2\u003e \u003cp\u003eFlies (35 flies/vial, 5 replicates per group) were collected after anesthetisation, and placed into vials containing 4.9g of diet prepared as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The rate of survival was derived from data analysis of the daily records of the flies\u0026rsquo; mortality. Also, the eclosion rate of \u003cem\u003eD. melanogaster\u003c/em\u003e offspring after exposure of the parent flies to copper ions and esculentin-2CHA(GA30) was evaluated as previously described\u003csup\u003e9,14\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of samples for biochemical assays\u003c/h2\u003e \u003cp\u003eAt the end of the treatment period treated flies (5 replicates per group) were anesthetised, collected, weighed, homogenized in 0.1 M phosphate buffer (pH 7.4) at a ratio of 1 mg:10\u0026micro;l of buffer. The homogenate was centrifuged at 4000 x g for 10 min at 4\u003csup\u003eo\u003c/sup\u003eC in a Mikro 220R centrifuge (Tuttlingen, Germany). Subsequently, supernatants were retrieved and stored at -20\u003csup\u003eo\u003c/sup\u003eC freezer until used for biochemical assays. All experiments were carried out in duplicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical assays\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of protein concentrations\u003c/h2\u003e \u003cp\u003eTotal protein concentration was measured as previously described by Adesanoye et al.\u003csup\u003e13\u003c/sup\u003e. This method was a slight modification of the protocol described by Lowry \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e48\u003c/sup\u003e. Briefly, fly homogenates (25 \u0026micro;l) were diluted ten times using distilled water. The diluted sample was subsequently mixed with Lowry\u0026rsquo;s reagent (400 \u0026micro;l). The mixture was then incubated at room temperature for 15 min. After the incubation period, the sample was diluted at a ratio of 1:5 with Folin Ciocalteau solution. The mixture was left at room temperature for 20 min. Absorbance readings were taken at 660nm. A standard curve made with graded concentrations of bovine serum albumin (BSA) was used for the interpolation of protein concentrations.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of nitric oxide (NO) levels\u003c/h2\u003e \u003cp\u003eThe concentration of nitrite in the supernatant was measured as described by Yao et al\u003csup\u003e49\u003c/sup\u003e. Briefly, samples (200\u0026micro;l) were incubated with Griess reagent (200\u0026micro;l) in the dark at room temperature for 20 min. The absorbance was read at 550nm. Nitrite concentrations were extrapolated from a standard curve constructed using known concentration of sodium nitrite.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels\u003c/h2\u003e \u003cp\u003eHydrogen peroxide generation was determined as described by Wolff\u003csup\u003e50\u003c/sup\u003e. Aliquots of homogenates (10\u0026micro;l) were mixed with 290\u0026micro;l of FOX reagent (10 ml of Xylenol Orange\u0026thinsp;+\u0026thinsp;10 ml of sorbitol\u0026thinsp;+\u0026thinsp;50 ml of AFS (Ammonium ferrous Sulfate) and 30 ml of distilled water. The reaction mixture was incubated at room temperature for 30 mins and the absorbance was read at 560nm. The concentration generated was extrapolated from the standard curve constructed using commercially available hydrogen peroxide.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of lipid peroxidation (LPO)\u003c/h2\u003e \u003cp\u003eLipid peroxidation was determined by the formation of thiobarbituric acid (TBA) reactive substances (TBARS) according to Varshney and Kale\u003csup\u003e51\u003c/sup\u003e method. The reaction medium contained 200 \u0026micro;l of trichloroacetic acid, 200 \u0026micro;l of thiobarbituric acid, and 100\u0026micro;l of fly homogenates. The mixture was incubated at 95\u003csup\u003eo\u003c/sup\u003eC for 1h. After cooling to room temperature, the mixture was centrifuged at 8000 x g for 10 min. The absorbance of aliquots of supernatant (300\u0026micro;l) was read at 532nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of protein carbonylation (PCO)\u003c/h2\u003e \u003cp\u003eThe spectrophotometric method described by Augustyniak \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e52\u003c/sup\u003e was used for the determination of absolute carbonyl levels. Homogenates (200\u0026micro;l) were mixed with equal volume of 2,4 dinitrophenylhydrazine (dissolved in 2.5M HCl). The mixture was vortexed and placed in the dark for 20 mins. Subsequently, 100 \u0026micro;l of 50% (w/v) Trichloroacetic acid was added to precipitate out the proteins. The mixture was incubated for 15 mins at -20\u003csup\u003eo\u003c/sup\u003eC and centrifuged at 4\u003csup\u003eo\u003c/sup\u003eC for 10 mins at 9000 rpm. The supernatant was discarded, and the pellet was washed twice in ice-cold 250 \u0026micro;l ethanol/ethyl acetate (1:1). The washed pellet was redissolved in 800\u0026micro;l of 6M guanidine hydrochloride. The absorbance was measured in the supernatant at 370nm, and carbonyl content was calculated.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of catalase (CAT) activity\u003c/h2\u003e \u003cp\u003eThe method of Aebi\u003csup\u003e53\u003c/sup\u003e was used in determining catalase activity. The reaction mixture consisted of 50mM potassium phosphate buffer (pH 7.0), 300 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and sample (1:50 dilution). The loss in absorbance of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was monitored for 2 min at 240 nm and thereafter used to calculate catalase activity expressed as \u0026micro;mol of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumed per minute per milligram of protein.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of glutathione-s-transferase (GST) activity\u003c/h2\u003e \u003cp\u003eGST activity was determined as described by Habig et al.\u003csup\u003e54\u003c/sup\u003e with 1-chloro-2,4-dinitrobenzene (CDNB) used as the substrate. Homogenates (20\u0026micro;l) were added to a reagent mixture (270\u0026micro;l) made with 20ml of 0.25M potassium phosphate buffer (pH 7.0) containing EDTA at a final concentration of 2.5 mM, 10.5ml of distilled water and 500\u0026micro;l of 0.1 M GSH at 25\u0026deg;C), and 10 \u0026micro;l of 25 mM 2,4, dichlorobenzene (DCNB). Absorbance was measured at 340nm at 30s intervals for 2 min.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eDetermination of non-protein thiol (NPSHs) level\u003c/h2\u003e \u003cp\u003eLevels of non-protein thiols (reduced gluthathione) were measured as described by Beutler \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e55\u003c/sup\u003e. Briefly, 50\u0026micro;l of trichloroacetic acid was mixed with 50\u0026micro;l of sample. The mixture was incubated at 4\u003csup\u003eo\u003c/sup\u003eC for 1 h prior to being centrifuged at 5000 x g for 10 minutes at 4\u003csup\u003eo\u003c/sup\u003eC. Subsequently, 50\u0026micro;l of the supernatant was mixed with 250\u0026micro;l of 0.1M Phosphate buffer (pH 7.4) and 50\u0026micro;l of Ellman\u0026rsquo;s reagent (5,5\u0026prime;-dithiobis-(2-nitrobenzoic acid, DTNB). Absorbance was measured at 412 nm using the microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eBehavioral /Neurotransmission Assay\u003c/h2\u003e \u003cdiv id=\"Sec27\" class=\"Section4\"\u003e \u003ch2\u003eDetermination of negative geotaxis\u003c/h2\u003e \u003cp\u003eThis assay was used to investigate the effects of Cu\u003csup\u003e2+\u003c/sup\u003e and esculentin-2CHa(GA30) on the climbing rate of the flies. This was performed as previously described by Abolaji \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e9\u003c/sup\u003e. Briefly, 30 flies per vial were treated and monitored for 5 days. After treatment, the flies were immobilized under mild ice. Anesthetized flies were placed (according to their groups) in labeled vertical glass columns (length 15cm, diameter 1.5cm). After recovery from the ice exposure, glass columns were gently tapped so that flies return to the bottom of the column. The number of flies that climbed up to the 6cm mark of the column within 6 seconds as well as those that were below this mark after the stipulated time were recorded. This procedure was repeated three times at 1min interval, and the climbing rate was calculated in percentage with respect to the total number of flies.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of acetylcholinesterase (AChE) activity\u003c/h2\u003e \u003cp\u003eAcetylcholinesterase activity was assayed as described by Ellman et al.\u003csup\u003e56\u003c/sup\u003e. Briefly, fly homogenates (10\u0026micro;l) were added to a reaction mixture containing 120\u0026micro;l of 0.1M potassium phosphate buffer (pH 7.4), 40\u0026micro;l of 10mM DTNB, 40\u0026micro;l of 8mM acetylthiocholine (the initiator) and 120\u0026micro;l of water. The reaction was monitored for 2 min (30 s intervals) at 412 nm. Data were calculated against reagent and sample blanks. The enzyme activity was estimated as \u0026micro;mol of acetylthiocholine hydrolyzed/minute/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of monoamine oxidase (MAO) activity\u003c/h2\u003e \u003cp\u003eMAO activity was quantified using the method described by Tipton et al.\u003csup\u003e57\u003c/sup\u003e with slight modification. In a 2 ml Eppendorf tube, 80\u0026micro;l of 0.1M phosphate Buffer (pH 7.4), 260 \u0026micro;l of distilled H\u003csub\u003e2\u003c/sub\u003eO, 20 \u0026micro;l of 2.5mM benzylamine hydrochloride, and 40 \u0026micro;l of sample were mixed. The mixture was incubated for 30 mins at 25\u003csup\u003eo\u003c/sup\u003eC. Afterward, 200 \u0026micro;l of perchloric acid was added to terminate the enzyme\u0026rsquo;s activity. The resulting solution was centrifuged at 1500 rpm for 10 mins. Absorbance was measured at 280nm.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistology\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eD. melanogaster\u003c/em\u003e in control and each treatment group after five (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) days of treatment were fixed in 150\u0026micro;l of diluted Bouin solution (1 ml of Bouin\u0026thinsp;+\u0026thinsp;10 ml of 0.1M PB) for 24 hours. Afterward, tflies were rinsed with 0.1M phosphate buffer (pH 7.4), until the yellow coloration of the Bouin solution was completely removed. Subsequently, rinsed flies were fixed in a solution of 10% Phosphate Buffer/Formalin. The flies were paraffinized and processed for hematoxylin and eosin (H\u0026amp;E) staining. An optic light microscope was used to view the brain.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error of Mean (SEM). Treated and control groups were compared using one-way ANOVA. Significant differences between groups were detected using Turkeys Post-hoc test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All analyses were carried out using Graph Pad Prism Version 6.0 software for Windows.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received for this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORSHIP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOLU, OOO, and AOA\u003c/strong\u003e contributed to the conception and design of the study, data interpretation and the preparation of manuscript. \u003cstrong\u003eOLU, AO, AAF, OOO\u003c/strong\u003e contributed to data collection and analysis. All authors approved the final manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSingh, R., Gautam, N., Mishra, A. and Gupta, R., 2011. Heavy metals and living systems: An overview. Indian journal of pharmacology, \u003cem\u003e43\u003c/em\u003e(3), pp.246\u0026ndash;253.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAguilar-Martinez, P., Grandchamp, B., Cunat, S., Cadet, E., Blanc, F., Nourrit, M., Lassoued, K., Schved, J.F. and Rochette, J., 2011. Iron overload in HFE C282Y heterozygotes at first genetic testing: a strategy for identifying rare HFE variants. \u003cem\u003ehaematologica\u003c/em\u003e, \u003cem\u003e96\u003c/em\u003e(4), p.507.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurutas, E.B., 2015. The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state. 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H., Calder\u0026oacute;n Guzm\u0026aacute;n, D., Hern\u0026aacute;ndez Garc\u0026iacute;a, E. and Barrag\u0026aacute;n Mej\u0026iacute;a, G., 2016. The role of dopamine and its dysfunction as a consequence of oxidative stress. \u003cem\u003eOxidative medicine and cellular longevity\u003c/em\u003e, \u003cem\u003e2016\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones, D.N. and Raghanti, M.A., 2021. The role of monoamine oxidase enzymes in the pathophysiology of neurological disorders. \u003cem\u003eJournal of Chemical Neuroanatomy\u003c/em\u003e, \u003cem\u003e114\u003c/em\u003e, p.101957\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J biol Chem, \u003cem\u003e193\u003c/em\u003e(1), pp.265\u0026ndash;275.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYao, D., Vlessidis, A.G. and Evmiridis, N.P., 2004. 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Redox biology, \u003cem\u003e4\u003c/em\u003e, pp.149\u0026ndash;157\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAebi, H., 1984. [13] Catalase in vitro. In \u003cem\u003eMethods in enzymology\u003c/em\u003e (Vol. 105, pp. 121\u0026ndash;126). Academic press.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabig, W.H., Pabst, M.J. and Jakoby, W.B., 1974. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. Journal of biological Chemistry, \u003cem\u003e249\u003c/em\u003e(22), pp.7130\u0026ndash;7139\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeutler, E., Duron, O. and Kelly, B.M., 1963. Improved method for the determination of blood glutathione. The Journal of Laboratory and Clinical Medicine, \u003cem\u003e61\u003c/em\u003e, pp.882\u0026ndash;888\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEllman, G.L., Courtney, K.D., Andres Jr, V. and Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical pharmacology, \u003cem\u003e7\u003c/em\u003e(2), pp.88\u0026ndash;95\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTipton, K.F., Davey, G. and Motherway, M., 2000. Monoamine oxidase assays. Current protocols in pharmacology, \u003cem\u003e9\u003c/em\u003e(1), pp.3\u0026ndash;6\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Copper toxicity, esculentin-2CHa-(GA30), amphibian skin peptides, neurodegeneration, neuroprotective effects, Drosophila melanogaster","lastPublishedDoi":"10.21203/rs.3.rs-4368804/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4368804/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExcess copper ion (Cu\u003csup\u003e2+\u003c/sup\u003e) has been implicated in various pathological conditions involving oxidative stress and inflammation. This study investigated neuroprotective effects of esculentin-2CHa-(GA30) on copper-induced toxicity in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. Flies were treated with esculentin-2CHa (5.0 and 7.5 \u0026micro;M/kg diet) and/or Cu\u003csup\u003e2+\u003c/sup\u003e (1mM) orally for 5 days. Effects of esculetin-2CHa-(GA30) on markers of redox-antioxidant status and neuro-behavioural activities were assessed. Esculetin-2CHa-(GA30) did not affect survival rate but reversed the effect of copper on eclosion rate. Esculetin-2CHa-(GA30) dose-dependently mitigated Cu\u003csup\u003e2+\u003c/sup\u003e-induced elevation of hydrogen peroxide (15.1\u0026ndash;15.8%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), thiobarbituric reactive substance (37.2\u0026ndash;55.1%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u0026ndash;0.001) and protein carbonyl (20.7\u0026ndash;63.8%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u0026ndash;0.001). Esculetin-2CHa-(GA30) ameliorated Cu\u003csup\u003e2+\u003c/sup\u003e-induced inhibition of catalase (1.5\u0026ndash;1.7-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u0026ndash;0.001), glutathione S-transferase activities (1.5\u0026ndash;2.1-fold, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u0026ndash;0.001) and decline in non-protein thiols levels (13.6\u0026ndash;27.7%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Esculetin-2CHa-(GA30) reduced Cu\u003csup\u003e2+\u0026minus;\u003c/sup\u003einduced elevation of monoamine oxidase (21.7\u0026ndash;39.7%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u0026ndash;0.01) and acetylcholinesterase (40.1\u0026ndash;55.9%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u0026ndash;0.001) activities. Copper-induced impaired locomotor activities were dose-dependently improved in esculentin-2CH-(GA30)-treated flies (21.4%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and 72.1%, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Histological assessments indicated the ability of esculentin-2CHa-(GA30) to sequester Cu\u003csup\u003e2+\u003c/sup\u003e in the microglia. In conclusion, esculentin-2CHa-(GA30) exhibited its neuroprotective effects through improved balance of redox status and associated behavioural characteristics. Further studies to delineate molecular mechanisms underlying observed effects would be required.\u003c/p\u003e","manuscriptTitle":"Esculentin-2CHa (GA30) mitigates copper-induced redox imbalance and behavioural deficit in Drosophila melanogaster","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-14 06:25:44","doi":"10.21203/rs.3.rs-4368804/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9edcc622-c630-4997-bdce-23eb7aaf73f6","owner":[],"postedDate":"May 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31855600,"name":"Biological sciences/Biochemistry"},{"id":31855601,"name":"Biological sciences/Developmental biology"},{"id":31855602,"name":"Biological sciences/Drug discovery"},{"id":31855603,"name":"Biological sciences/Neuroscience"},{"id":31855604,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2024-08-09T14:29:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-14 06:25:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4368804","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4368804","identity":"rs-4368804","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}