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

doi:10.21203/rs.3.rs-4368804/v1

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

https://doi.org/10.21203/rs.3.rs-4368804/v1

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Excess copper ion (Cu²⁺) 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 Cu²⁺ (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 Cu²⁺-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 Cu²⁺-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 Cu²⁺⁻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 Cu²⁺ 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

Copper (Cu) is a transition metal that is essential for many cellular processes¹. 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 homeostasis². 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 tau⁸. Similarly, previous studies revealed that excessive copper intake affects cognition and plays a role in the pathogenesis of AD, Parkinson Disease, and amyotrophic lateral sclerosis⁹. 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 activity¹⁰. 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 chiricahuensis¹¹. The frog skin host-defense peptide, esculentin-2CHa has been shown to display multifunctional effects including antimicrobial, antitumor, and immunomodulatory properties¹¹. Esculentin-2CHa(GA30) is the truncated version of the original peptide, lacking the last seven amino acid residues¹². Antidiabetic¹² and antioxidative effects¹³ 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 humans¹⁴. 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.

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 Cu²⁺ in the absence or presence of esculentin-2CHa(GA30) die over a 5-day period is presented in Figs. 1A and 1B. 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. 1C). 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 Cu²⁺ 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 Cu²⁺ on eclosion rates was more pronounced (Fig. 1D and 1E). The rate of emergence of new flies in Cu²⁺ 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 Cu²⁺ and by 1.1-fold (ns) in the presence of Cu²⁺ compared to control flies. The effect observed in the group treated with peptide (7.5µM/kg diet) and Cu²⁺ represent a 1.7-fold (P < 0.05) increase compared to flies treated with Cu²⁺ 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 Cu²⁺ 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 Cu²⁺ 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 Cu²⁺ on the number of emerging flies was also completely inhibited at this peptide concentration (Fig. 1F).

Effects of esculentin-2CHa(GA30) on H ₂ O ₂ and NO levels in Cu²⁺-treated Drosophila melanogaster

H₂O₂ and NO levels were assessed as markers of oxidative stress in this study. Results obtained indicate that treatment of flies with Cu²⁺ led to 16.2% (P < 0.05 increase in H₂O₂ levels (Fig. 2A) compared to control flies. Unlike Cu²⁺, the treatment of flies with esculentin-2CHa(GA30) alone at all concentrations tested did not produce significant increase in H₂O₂ levels. However, the elevation of H₂O₂ levels produced by Cu²⁺ 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. 2A). NO levels in control flies and flies treated with esculentin-2CHa(GA30) were similar. However, treated with Cu²⁺ increased NO levels by 36.4% (P < 0.05). The combination of Cu²⁺ with esculentin-2CHa(GA30) did not reduce the elevation in NO levels produced by Cu²⁺ (Fig. 2B).

Effects of esculentin-2CHa(GA30) on lipid peroxidation and protein carbonylation in Cu²⁺-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 Cu²⁺ only (Fig. 3A). The treatment of flies with esculentin-2CHa(GA30) at both dosages used in this study significantly reduced basal and Cu²⁺-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 Cu²⁺-induced elevation of TBARS by 37.2% (P < 0.01). A more significant reduction in basal (40.8%, P < 0.01) and Cu²⁺-induced (55.1%, P < 0.001) TBARS levels was observed in flies treated with esculentin-2CHa(GA30) at 7.5µM/kg diet (Fig. 3A). Similarly, protein carbonyl levels in Cu²⁺-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 Cu²⁺-induced (47.0%, P < 0.01) levels of protein carbonyls. Similarly, reduced basal (33.6%, P < 0.01) and Cu²⁺-induced (63.8%, P < 0.001) levels of protein carbonyls were observed in flies treated with the peptide at 7.5µM/kg diet (Fig. 3B).

Effects of Esculentin-2CHa(GA30) on activities of catalase and glutathione-S-transferase in Cu²⁺-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. 4A). 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 Cu²⁺ resulted in the inhibition of Cu²⁺-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. 4A). 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. 4B).

Effects of esculentin-2CHa(GA30) on levels of non-protein thiols and total thiols in Cu²⁺-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. 4C). 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 Cu²⁺ 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 Cu²⁺-treated flies and at a peptide concentration of 7.5µM/kg diet, the deleterious effect of Cu²⁺ was completely inhibited (Fig. 4D). 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. 4D). 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 Cu²⁺, 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. 5A). However, following the exposure of flies to Cu²⁺, only 52.7 ± 4.1% of flies had the ability for negative geotaxis. Treatment of flies exposed to Cu²⁺ with esculentin-2CHa(GA30) at 5.0µM/kg diet completely prevented the effect of Cu²⁺ 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 Cu²⁺ alone (Fig. 5A).

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 Cu²⁺, increased monoamine oxidase activities by 1.4-fold (P < 0.001). However, the treatment of flies exposed to Cu²⁺ with esculentin-2CHa(GA30) at 5.0 and 7.5 µM/kg diet completely inhibited the effect of Cu²⁺ and reduced monoamine oxidase by 21.7% (P < 0.05) and 39.7% (P < 0.01) respectively when compared to control flies (Fig. 5B). 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. 5C). Increased enzyme activities (1.9-fold, P < 0.001) was observed in flies exposed to Cu²⁺. The treatment of Cu²⁺-exposed flies with esculentin-2CHa(GA30) reduced the effect of Cu²⁺ 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 Cu²⁺. A moderate and significant repopulation of neurons in the grey matter of the lobular of flies co-treated with the peptide and Cu²⁺ 5.0µM and 7.5 µM respectively was observed was observed (Fig. 6).

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 reported⁹. 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 neurons¹⁸. 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 reported⁹. 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 study¹³.

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 laboratory⁹. 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 radicals²¹. 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 cell²². In this study, the exposure of flies to Cu²⁺ led to elevated levels of H₂O₂ level when compared to the control. This observation is consistent with the previous report which indicated that copper interacts with hydrogen peroxide (H₂O₂) 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 flies⁹. 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 reported²³. Elevated levels of cellular markers of lipid peroxide generation in liver homogenates of rats intoxicated with copper has also been reported²⁴. 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 suggested¹³. 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 study³¹. These metabolites diffuse from the membrane into the cytoplasm and nucleus where they covalently bind to cysteine, histidine, or lysine residues of proteins³². Carbonylation alters protein function, leading to deleterious intermolecular cross-links and aggregates that preclude their degradation by intracellular proteases³³. 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 Sclerosis³⁴. 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 damage³⁵. Actions of these enzymes are often supported by defensive mechanisms mediated by cellular constituents such as free amino acids, glutathione, and phenolic compounds³⁶. 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 cell³⁸ 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 rats³⁹, mice⁴⁰, cultured cells⁴¹, and Drosophila⁹ 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 brain⁴². 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 choline⁴³, and imbalances in acetylcholine metabolism has been linked with chronic conditions like Alzheimer’s disease and Parkinsonism⁴⁴. These studies also highlight a significant role for AChE inhibitors in the management of neurodegenerative disorders⁴⁴. In this study, esculentin-2CHa(GA30) significantly inhibited Cu²⁺-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 function⁴⁵. 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 learning⁴⁶. Consistent with these, monoamine oxidase inhibitors prevent neurotransmitter loss and preserve normal function of the brain⁴⁷. 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 Cu²⁺ 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.

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 described¹³. 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.

Cu²⁺ exposure and Esculentin-2CHa(GA30) treatment

Based on previous studies in our laboratory, a dose of 1mM Cu²⁺ derived from copper sulfate pentahydrate (CuSO₄.5H₂O)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 Cu²⁺-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 + CuSO₄ (1mmol/kg diet) Diet + CuSO₄ (1mmol/kg diet) + Esculenthin-2CHa (5.0 µmol/kg diet) Diet + CuSO₄ (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^oC in a Mikro 220R centrifuge (Tuttlingen, Germany). Subsequently, supernatants were retrieved and stored at -20^oC 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 al⁴⁹. 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 (H₂O₂) levels

Hydrogen peroxide generation was determined as described by Wolff⁵⁰. 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 Kale⁵¹ 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^oC 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^oC and centrifuged at 4^oC 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 Aebi⁵³ was used in determining catalase activity. The reaction mixture consisted of 50mM potassium phosphate buffer (pH 7.0), 300 mM H₂O₂ and sample (1:50 dilution). The loss in absorbance of H₂O₂ was monitored for 2 min at 240 nm and thereafter used to calculate catalase activity expressed as µmol of H₂O₂ 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^oC for 1 h prior to being centrifuged at 5000 x g for 10 minutes at 4^oC. 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 Cu²⁺ 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 H₂O, 20 µl of 2.5mM benzylamine hydrochloride, and 40 µl of sample were mixed. The mixture was incubated for 30 mins at 25^oC. 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.

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.

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

Status:

Version 1

Abstract

INTRODUCTION

RESULTS

Effect of esculentin-2CHa(GA30) on survival and eclosion rates in Drosophila melanogaster

Effects of esculentin-2CHa(GA30) on lipid peroxidation and protein carbonylation in Cu²⁺-treated Drosophila melanogaster

Effects of Esculentin-2CHa(GA30) on activities of catalase and glutathione-S-transferase in Cu²⁺-treated Drosophila melanogaster

Effects of esculentin-2CHa(GA30) on levels of non-protein thiols and total thiols in Cu²⁺-treated Drosophila melanogaster

Effect of esculentin-2CHa(GA30) on behavioural and neurotransmission markers in Drosophila melanogaster

Copper-chelating effects of esculentin-2CHa(GA30) in the brain of Drosophila melanogaster

DISCUSSION

MATERIALS AND METHODS

Chemicals

Peptide synthesis and purification

Drosophila melanogaster stock and culture

Cu²⁺ exposure and Esculentin-2CHa(GA30) treatment

Survival and emergence rate of flies

Preparation of samples for biochemical assays

Biochemical assays

Determination of protein concentrations

Determination of nitric oxide (NO) levels

Determination of hydrogen peroxide (H₂O₂) levels

Measurement of lipid peroxidation (LPO)

Measurement of protein carbonylation (PCO)

Determination of catalase (CAT) activity

Determination of glutathione-s-transferase (GST) activity

Determination of non-protein thiol (NPSHs) level

Behavioral /Neurotransmission Assay

Determination of negative geotaxis

Determination of acetylcholinesterase (AChE) activity

Determination of monoamine oxidase (MAO) activity

Histology

Statistical analysis

Declarations

References

Additional Declarations

Status:

Version 1

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

Status:

Version 1

Abstract

INTRODUCTION

RESULTS

Effect of esculentin-2CHa(GA30) on survival and eclosion rates in Drosophila melanogaster

Effects of esculentin-2CHa(GA30) on lipid peroxidation and protein carbonylation in Cu2+-treated Drosophila melanogaster

Effects of Esculentin-2CHa(GA30) on activities of catalase and glutathione-S-transferase in Cu2+-treated Drosophila melanogaster

Effects of esculentin-2CHa(GA30) on levels of non-protein thiols and total thiols in Cu2+-treated Drosophila melanogaster

Effect of esculentin-2CHa(GA30) on behavioural and neurotransmission markers in Drosophila melanogaster

Copper-chelating effects of esculentin-2CHa(GA30) in the brain of Drosophila melanogaster

DISCUSSION

MATERIALS AND METHODS

Chemicals

Peptide synthesis and purification

Drosophila melanogaster stock and culture

Cu2+ exposure and Esculentin-2CHa(GA30) treatment

Survival and emergence rate of flies

Preparation of samples for biochemical assays

Biochemical assays

Determination of protein concentrations

Determination of nitric oxide (NO) levels

Determination of hydrogen peroxide (H2O2) levels

Measurement of lipid peroxidation (LPO)

Measurement of protein carbonylation (PCO)

Determination of catalase (CAT) activity

Determination of glutathione-s-transferase (GST) activity

Determination of non-protein thiol (NPSHs) level

Behavioral /Neurotransmission Assay

Determination of negative geotaxis

Determination of acetylcholinesterase (AChE) activity

Determination of monoamine oxidase (MAO) activity

Histology

Statistical analysis

Declarations

References

Additional Declarations

Status:

Version 1

Effects of esculentin-2CHa(GA30) on lipid peroxidation and protein carbonylation in Cu²⁺-treated Drosophila melanogaster

Effects of Esculentin-2CHa(GA30) on activities of catalase and glutathione-S-transferase in Cu²⁺-treated Drosophila melanogaster

Effects of esculentin-2CHa(GA30) on levels of non-protein thiols and total thiols in Cu²⁺-treated Drosophila melanogaster

Cu²⁺ exposure and Esculentin-2CHa(GA30) treatment

Determination of hydrogen peroxide (H₂O₂) levels