Adult Life Phase-Specific Dopaminergic Neuroprotective Efficacy of Curcumin is through Variant Modulation of Brain Dopamine Metabolism: Insights from ALSS Drosophila Model of Parkinson’s Disease

Adult Life Phase-Specific Dopaminergic Neuroprotective Efficacy of Curcumin is through Variant Modulation of Brain Dopamine Metabolism: Insights from ALSS Drosophila Model of Parkinson’s Disease | 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 Adult Life Phase-Specific Dopaminergic Neuroprotective Efficacy of Curcumin is through Variant Modulation of Brain Dopamine Metabolism: Insights from ALSS Drosophila Model of Parkinson’s Disease Abhik Das, Rahul Chaurasia, Priyanka Modi, Mohamad Ayajuddin, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4700590/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 Epidemiological studies suggest a strong link between exposure to environmental toxins and onset of Parkinson’s disease (PD). Our laboratory has developed an adult life stage-specific (ALSS) Drosophila model of sporadic PD which is critical to screen small molecules and identify molecular targets of dopaminergic (DAergic) neuroprotection for late-onset neurodegenerative diseases (NDD) such as PD. Nutraceutical curcumin (CU) has been a time-tested ingredient in the Asian kitchen, traditional medicine and has been employed in 450 clinical trials. Exposure to Paraquat (PQ) induces mobility defects in the health stage (point of time when there is no natural diseases) and transition stage (time period wherein about 10% deaths occur) of adult Drosophila; whereas CU ameliorates the deficits only during early health span but fails during late health and transition phases. Probing the whole fly brain using anti-tyrosine hydroxylase (anti-TH) antibodies, for PQ-mediated dopamine (DA) neurodegeneration illustrates that it does not cause loss of DA neurons per se . However, it leads to DA “neuronal dysfunction” (diminished levels of rate-limiting enzyme in dopamine synthesis- TH) and CU rescues the neuronal dysfunction only during the early health span but fails to mitigate the DA neuronal pathology during the transition phase of adult life. Genotropic nutraceutical CU replenishes the diminished levels of brain-specific DA and its metabolites DOPAC and HVA during the adult health phase (HP) and fails to do so in the adult transition phase (TP), suggesting its life phase-specific dopaminergic neuroprotective efficacy is mediated through differential modulation of perturbations in brain dopamine metabolism. The present study suggests the limitation of CU as a therapeutic strategy for PD and emphasizes the necessity and importance of screening putative neuroprotective small molecules for late onset NDD such as PD in life phase matched animal models during which the disease sets in. Biological sciences/Biochemistry Biological sciences/Cell biology Biological sciences/Developmental biology Biological sciences/Neuroscience Biological sciences/Zoology Curcumin Dopamine Drosophila Health phase Parkinson’s disease Paraquat Transition stage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Parkinson’s disease (PD) is a movement disorder prevalent in 1% of the population worldwide. Epidemiological studies have shown a strong correlation between the onset of PD and exposure to environmental neurotoxins such as paraquat (PQ) and rotenone ( 1 – 4 ). The pathogenesis of PD is still yet to be fully elucidated due to the multifactorial nature of the disease. The underlying mechanisms of the dopaminergic (DAergic) neuronal loss are primarily driven by the generation of reactive oxygen species (ROS), decrease in antioxidant enzyme levels, neuroinflammation, mitochondrial dysfunction, and ER stress, leading to a cascade of molecular cross-talks that result in the initiation of neuronal dysfunction ( 5 , 6 ). Several animal models of PD have been developed using the herbicide PQ. It has been used in experimental studies focusing on its pathological effects on the brain, heart, lungs, kidneys, liver, and muscle due to systemic toxicity and fatality after acute exposure. The interest in using PQ as a neurotoxin to model PD started since its discovery due to its similarity in terms of its molecular structure and biochemistry with 1-methyl-4-phenylpyridinium (MPP + ), the active metabolite of MPTP, a neurotoxin that can penetrate the blood brain barrier (BBB) and induce PD-like features in animal models and humans ( 7 , 8 ). The differential expression of multiple genes during the different life stages signifies the process of aging ( 9 , 10 ). The adult life stages of Drosophila are categorized into health (no natural death occurs), transition (slight decline in the mortality curve showing 10% death), and senescence stage (steady decline in mortality curve, represented by the window between the end of the transition phase till maximum life span of the fly) ( 11 ). These life stages of Drosophila , are characterized by different patterns of gene expression ( 12 , 13 , 10 ), like humans ( 14 ). The transcriptomic analysis of the gene expression profiles in Drosophila melanogaster has acknowledged 1184 genes with prominent differences in the expression levels between young and old age groups ( 9 ). A large number of adult life stage-associated pathways independently influence common and unique complex biological processes of Drosophila life stages ( 15 ). All these studies emphasize the importance and necessity of developing life stage-specific animal models for late-onset NDD such as PD. Studies in the field of polyphenols and their potential benefits in modern medicine for their positive outcome on human health are becoming very common. Natural products possessing diverse biological activities and drug-like properties are important resources for treating human diseases ( 16 ). Researchers have suggested the efficacy of natural products by demonstrating their efficacy in modulating biochemical markers, anti-oxidant enzymes and phenotypes associated with the disease in different animal models. Their varied natural role in organisms overlays the basis for their therapeutic prospect in presently non-treatable neurodegenerative disorders including PD. Hence, natural products present in our daily diet that could promote healthy aging are intensively studied. Curcumin (CU) is an extensively investigated phytochemical with over 20000 PUBMED citations in the last two decades (among these, 303 studies are on PD) and is proven to possess powerful anti-inflammation, anti-viral, anti-oxidant, anti-biotic, anti-depressant, anti-arthritic, wound healing properties, anticancer, and anti-neurodegenerative characteristics ( 17 , 18 ). It has been employed in 316 clinical trials (116 trials in the USA only) for different human diseases (ClinicalTrial.gov; viewed on 25/11/2023). The therapeutic efficacy of CU has been demonstrated in various diseases such as cancer ( 19 , 20 ), cardiovascular diseases ( 21 ), and various neurological disorders such as ALS ( 22 ), anxiety ( 23 ), depression ( 24 ), AD ( 25 , 26 ), Schizophrenia ( 27 ) and PD ( 28 , 29 ). It has been reported to be a strong forager of a range of ROS, including hydroxyl radicals, superoxide anion radicals, and protection of oxidative damage to kidney cells by suppressing lipid degradation, lipid peroxidation and cellular breakdown ( 30 ). Randomized clinical trials comprising 631 patients with various diseases have shown the beneficial role of CU/turmeric ( 31 ). It is shown to be beneficial in skin diseases such as psoriasis, pruritic skin lesions, radiation dermatitis, vitiligo, etc. ( 32 ). CU is found to be beneficial in digestive disorders such as indigestion, heartburn, nausea, constipation, diarrhoea, abdominal pain, physical functioning, energy levels and sleep ( 33 ). CU is shown to have a beneficial role in several gynaecological disorders ( 34 ). CU is also shown to upregulate several genes in an in vitro human gingival fibroblast wound healing model suggesting its role as a therapeutic agent for gingival ulcers ( 35 ). Thus, CU is one such potential candidate that can be explored for a therapeutic approach to several human diseases including NDD, like PD. It may be noted that the investigation of different phases of life in Drosophila has shown that each life stage is distinguished by a diverse pattern of gene expression ( 10 , 36 ). This pattern is comparable to the corresponding life phase in mice, flies and humans. However, several studies have shown the neuroprotective effects of CU by employing young animal models of the adult health phase ( 37 – 40 ). Therefore, while screening nutraceuticals for their DAergic neuroprotective efficacy in animal models, it is important to follow the adult life stage/phase-specific studies for late-onset NDD such as PD ( 8 , 41 ). The studies concerning CU safety under various experimental conditions are under-reported. Reports suggesting that CU may cause toxicity under specific conditions are shown with the studies in mammalian cell lines on treatment with turmeric in which there is a dose and time-dependent induction of chromosome aberrations ( 42 ). Another study on the effect of CU on DNA has demonstrated that it induces DNA damage and chromosomal alterations ( 43 ). An experiment testing the chelator action of CU to cause iron deficiency in vivo showed that CU suppressed the production of hepcidin, a peptide that plays an essential role in the regulation and balancing of systemic iron. It causes iron insufficiency, leading to anemia in mice that are fed poor iron diets ( 44 ). CU confers negative activity of enzymes such as glutathione-S-transferase and UDP-glucuronosyl transferase apart from hampering the action of drug-metabolizing enzymes such as cytochrome P450 ( 45 ). Such action of CU may result in abnormal plasma flow of certain drugs that may lead to toxic action ( 46 ). Contradicting the reports that suggest beneficial properties of CU, a study suggested both advantages and disadvantages of CU in alcoholic liver injury. Concentration-dependent CU toxicity was reported in animal models in which CU was found to accelerate liver injury and liver cellular oedema ( 47 ). In a study conducted by our lab to assess the toxicity of CU, it was shown that CU induces dose-dependent lethality in which a concentration of 2.5 mM and above adversely affected the viability of Drosophila ( 8 ). Another study showed that a concentration of 1 mg/L aqueous extract of Curcuma longa induces an adverse effect on the viability of normal SH-SY5Y cells ( 48 ). Hence, the use of CU as a therapeutic agent must be moderated at an optimal dose to avoid its toxic side effects ( 49 ). Therefore, while developing a therapeutic approach for a disease, it is important to thoroughly validate the toxicity as well as beneficial activity of the drug/phytochemical per se in the model organism. Taking this important aspect into consideration, our laboratory employed a wide range of CU concentrations and assessed their possible toxicity in the adult life phase-specific Drosophila model and determined certain sub-lethal concentrations (508). These selected concentrations neither influence viability nor induce mobility defects in Drosophila and were employed for assessing the neuroprotective efficacy of CU in further studies ( 50 , 8 ). Our laboratory has previously demonstrated the adult life stage-specific neuroprotective efficacy of CU in a PQ-mediated Drosophila model of PD ( 8 ). Hence, in this study we made an effort to understand the neurophysiological and neurochemical aspects of adult life stage-specific (ALSS) neuroprotective efficacy of CU in the Drosophila model of sporadic PD. By intervention of CU (500 µM and 1 mM) as was demonstrated in Phom et al. , ( 8 ), in PD brain. We have demonstrated that time-tested nutraceutical CU has limitations to its therapeutic efficacy for late-onset NDD such as PD. The ALSS DAergic neuroprotective efficacy of CU is mediated through the prevention of “neuronal dysfunction” and differential regulation of brain dopamine (DA) metabolism. This knowledge would help to modulate existing curcumin/nutraceutical mediated therapeutic strategies and assist further in developing efficient healing approaches for late-onset NDD like PD. Materials and Methods Fly husbandry Fly was cultured according to the protocol described in Phom et al . ( 8 ). Oregon K (OK) (procured from National Drosophila Stock Center, Mysuru University, Mysuru, India) male flies of D. melanogaster was used in the present study. The flies were raised at 22 ± 1 0 C with 12 Hours (Hrs) light and dark cycle in a fly incubator (Percival, USA). The flies were fed with a culture medium composed of sucrose, yeast, agar-agar and propionic acid ( 8 ). For collecting the flies, they were mildly anesthetized with a few drops of diethyl ether. Only 25 flies were kept in each vial containing fresh media. The collected flies were transferred to a fresh media vial every third day. 4–5 day flies were used for further experiments while late health span and transition phase flies were kept transferring routinely for every 3rd day till they reached a specific life stage and then were used for experiments. Chemicals The required chemicals viz.,Sucrose (SRL, Maharashtra, India,Cat: 84973),Paraquat (PQ; methyl viologen dichloride hydrate,Sigma-Aldrich, St. Louis, MO, United States,Cat: 856177), Curcumin (CU; Sigma-Aldrich, St. Louis, MO, United States,Cat: 1386) and DMSO (Sigma-Aldrich, St. Louis, MO, United States, Cat: D8418) were used for feeding procedures. Standard dopamine (DA; Sigma-Aldrich, St. Louis, MO, United States,Cat: H8502) and its metabolites-3,4-Dihydroxyphenylacetic acid (DOPAC; Sigma-Aldrich, St. Louis, MO, United States, Cat: 11569) and Homovanilic acid (HVA; Sigma-Aldrich, St. Louis, MO, United States,Cat: 69673), Phosphate-buffered Saline (PBS; HiMedia,Maharashtra, India, India, Cat: ML023), Trichloro Acetic Acid (TCA, SRL, Maharashtra, India, Cat: 204842) were used for quantifying DA and metabolites. Paraformaldehyde (Sigma-Aldrich, St. Louis, MO, United States,Cat: I58127), Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States,Cat: T8787), Normal Goat Serum (NGS; Vector Labs, CA, United States,Cat: S1000), VECTASHIELD® mounting medium (Vector Labs, CA, United States,Cat:H1000), Rabbit anti-Tyrosine hydroxylase (anti-TH) polyclonal primary antibody (Millipore, MA, United States, Cat: Ab152) and Goat anti-rabbit IgG H&L(TRITC labelled) polyclonal secondary antibody (Abcam, MA, United States, Cat: Ab6718) were used for immunostaining. Treatment Protocol The fly treatment and CU intervention protocol follows that of Phom et al. , ( 8 ). Briefly, male flies were aged for 4 days (Health phase: HP) and 55 days (Transition phase: TP) feeding in sucrose-agar media. The flies were transferred to freshly prepared media every 3rd day while aging. The flies were then transferred to vials (30mm x100mm) containing disc of Whatman filter paper no. 1 saturated with 275µl of 5% sucrose, 10mM PQ in 5% sucrose, CU (500µM, 1mM) with PQ (10mM) and Curcumin in DMSO. At 24hrs of exposure, flies were frozen at -80˚C. For decapitation of head as required for HPLC, a chilled aluminium tray was positioned on the ice block and flies were placed on it. Using Carl Zeiss stereozoom (Stemi 305) microscope the flies were then dissected separating head from body with a sharp razor. Quantification of DAergic neurodegeneration and Tyrosine hydroxylase synthesis in whole fly brain using fluorescence microscopy Mounting of the whole fly brain for fluorescence microscopy (Carl Zeiss, Axio Imager M2, with ZEN 2012 SP2 software, Germany) was done as described in Ayajuddin et al. , ( 51 ). Elaborately, the brains of male Oregon K flies were fixed in 4% paraformaldehyde (PFA) containing 0.5% TritonX (TX)-100, at room temperature for 2 Hrs, and then washed five times afterevery 15 minutes (5 X 15 min) in phosphate-buffered saline (PBS) with 0.1% TX-100 (PBST), at room temperature (RT). Blocking was performed using PBS containing 0.5% TX-100 and 5% normal goat serum (NGS) for 120 minutes at room temperature (RT). Then, primary antibody (anti-TH) incubation was done for the brains with aratio of 1:250 for 72 Hrs at 4° C. The excess primary antibody was washed off from the brains for 5 X 15 minutes with PBST. Brains were then incubated with 1:250 dilution of secondary antibody (TRITC labelled) for 24 Hrs at RT under dark conditions. After thorough washing for 5 X 15 minutes in PBST to remove the excess secondary antibodies, brains were mounted in VECTASHIELD® mounting mediumthen topped with cover glass (Electron Microscopy Sciences,PA, USA), and image acquisition was done on the same day. The quantification of DAergic neurons and level of tyrosine hydroxylase (TH) protein synthesis was done as per Ayajuddin et al. , ( 51 ). Briefly, prepared/stained brains were viewed under a fluorescence microscope at a 40x. The image was scanned using a monochromatic camera witha Rhodamine filter using a red dot test for the visibility of neuron(s) and assessing saturation. Then, Z-stack programming with constant intervals was performed. For image processing, on the method column, image subset and maximum intensity projection (MIP) with X–Y Plane was created.From 3D scan images of Z- stack, PAL, PPL1, PPL2, PPM1/2, PPM3, (PAL- Protocerebral anterior lateral; PPL- Protocerebral posterior lateral; PPM- Protocerebral posterior medial) brain regions were selected. The images were enlarged to see clear neurites, then from graphics appropriate tools ‘draw spline contour’ were selected and a line was drawn around the neuron creating intensity sum in .xml format. The same process was followed for each neuron located in different clusters. The fly brain with the same orientation was carefully chosen for FI quantification. Protein Extraction from Drosophila brains From each of the treatments 50 fly heads were homogenized in 160ul of RIPA buffer (50mM Tris HCL, 1% Triton, 0.5% sodium deoxycholate, 150mM NaCl, 0.1% SDS, 2mM EDTA) with protease inhibitor cocktail. Homogenates were then sonicated for 20sec (with pulse of 10sec and amplitude at 30%) using Qsonica sonicators (from OHIO industries). The samples were centrifuged at 13,000rpm for 5min at 4⁰C. Supernatant was re-centrifuged at 13,000rpm for 5min at 4⁰C. Lysates were stored at -80⁰C until quantification was performed. Protein quantification Protein quantification was performed using the Bio-rad RCDC assay reagents (cat no. 500 − 0120). Bovine serum albumin at concentration of 1mg/ml (cat no. A-2153, Sigma) was used as the standard and 5ul of the extracted protein lysates were used for quantification of the samples. Absorbance was read at 750nm wavelength. Western blotting SDS-polyacrylamide gels were cast using the TGX stain-free fast cast acrylamide kit, 10% from Bio-Rad (cat no 161-0183TA) of 1.5mm thickness. Bio-rad proprietary method for western blotting Prepare resolving gel solution by mixing equal volumes of resolver A and resolver B solution (as described by the manufacturer). Use casting stand to stabilize the glass plates during casting. Add required volume of TEMED and freshly prepared 10% APS to the combined resolver and mix well. Steadily dispense the solution into the glass plates. Fill the cassette to 1cm below the bottom of the teeth of comb. Prepare stacking gel acrylamide solution by combining equal volumes of stacker A and stacker B solution (as described by the manufacturer). Add the required volume of TEMED and 10% APS to the stacker solution and mix well. Pipet the solution in the middle of the cassette, filling to the top of short plate. Apply slowly and steadily to prevent mixing with resolving solution. Align and insert the comb in the cassette. Allow the gel to polymerize for 30–45 min before electrophoresis. 40ug of each sample lysate were mixed with 20ul of sample buffer (0.5M Tris HCl pH6.8, 10%SDS, glycerol, 0.1% bromophenol blue, β mercaptoethanol) in a total volume of 40ul and denatured for 5min at 95⁰C. Bio-Rad prestained plus protein dual color standard (161–0374) was used as the protein ladder marker. A 10X stock of running buffer (Tris, Glycine, and SDS) was used for preparing 1X running buffer. The gel was run at a current of 20mAmp at room temperature using Bio-Rad powerpac basic power system. Before electro blotting, the stain free gels were scanned and activated (2.5min) using the Bio-Rad fluorescent documentation system. PVDF membrane (Bio-Rad 162–0174) was used after activation by methanol (Merck) before setting up the transfer sandwich. Transfer was carried out at a voltage of 90V for the duration of 90min using chilled 1X transfer buffer (Glycine, Tris and methanol) with continuous stirring using magnetic bead. To create a cold temperature condition the transfer tank placed inside a bucket filled with ice. Membrane blocking and Antibody treatment Post transfer the PVDF membrane was scanned using the Bio-Rad fluorescent documentation and incubatedin the blocking buffer of 5% BSA in 1X TBS-T (0.05%) for the duration of 90min at room temperature with gentle rocking. Rabbit polyclonal to Tyrosine Hydroxylase (ab152) was used in the dilution of 1:1000 and the membrane was incubated at 4⁰C for 48hrs. Post primary antibody incubation, the membrane was washed 3X in 1X TBS-T (0.05%) for 15min at room temperature. The secondary antibody Goat anti rabbit HRP (abcam 205718) was used in a dilution of 1:5000 and the membrane was incubated for 24hr at4⁰C. The membrane was washed 5X in 1XTBS-T (0.05%) for 15 min and developed using Clarity western ECL substrate (Bio-Rad 170–5060). Scanning was performed with Bio-Rad fluorescent documentation system. Data analysis data analysis was performed using ImageLab 5.2.1 version software Quantification of brain dopamine and its metabolites using High Performance Liquid Chromatography - Electro chemical detector (HPLC-ECD) Brain-specific DA and its metabolites were quantified using HPLC-ECD (HPLC-Thermo Scientific, Dionex Ultimate 3000) following the protocol described by Ayajuddin et al. , ( 51 ). Control group and PQ exposed group of flies were immediately frozen following 24 hrs of exposure. To avoid thawing of tissue and degradation of biomolecules, frozen flies were placed on ice tray containing chilled metal surface and 15 fly heads were decapitated quickly with a sharp scalpel. Head tissue homogenate was preparedin 300 µl of chilled PBS. Sonication of the homogenate was performed at 30 percent amplitude for 20 seconds with 5 seconds of interval. Followed by sonication homogenate was centrifuged at 6,000 rpm, 4 ◦ C for 10 minutes. After centrifugation, 50 µL of the supernatant was set aside for protein quantification. Rest of the supernatant was combined with 5% TCA (prepared in HPLC grade or enzyme free water) in a 1:1 ratio and kept in ice. Standard DA, DOPAC, HIAAand HVA were prepared in PBS, each having a concentration of 200 ng/ml. The standard solution was mixed with 5% TCA in a 1:1 ratio and kept in ice to prevent catecholamine degradation. For quantification, 50 µl of the tissue sample and 20 µl of the composite standard were loaded into the HPLC. MCM 15 cm X 4.6 mm, 5 µ C- 18 packed column (Thermo-Scientific, Waltham, USA, Cat: 70–0340) was used as the stationary phase for elution of the catecholamines, and MD-TM served as the mobile phase. To detect the catecholamines, the reduction and oxidation potentials within the twocells of primary ECD, were kept at -175 mV and + 225 mV respectively. The secondary ECD module acting as third cell also known as Omnicell, was set to + 500 mV in order to reduce background noise. Data was gathered at a rate of 5 Hz. Chromatogram analysis was done using Chromeleon®7 from Thermo-Scientific (Waltham, USA). Comparisons were made between sample and standard chromatograms for a catecholamine's retention time. To precisely pinpoint the peaks corresponding to DA, DOPAC, and HVA in the sample, 10 µL of the composite standard was added in sample and run through the HPLC once again. The spiked peaks according to the detection sequence in standard solution were recognized as the catecholamines of interest in sample. Quantification and normalization of catecholamines is described in Ayajuddin et al. , ( 51 ). In brief, ( 1 ). Concentration of a catecholamine is: C Std (ng/ml), ( 2 ). Area of a catecholamine in the composite standard chromatogram is: A Std, and injection volume of the composite standard solution is: I Std (µL), ( 3 ). Area of the catecholamine in the tissue extract chromatogram is: A Samp and the injectionvolume of the tissue extract is I Samp (µL), ( 4 ) Total number of fly heads for protein extraction: N, ( 5 ). The total protein concentration of the tissue extract is: P Samp (µg/µL). Calculation steps: The standard catecholamine concentration in I Std (µL) injection volume: (C Std X I Std )/1,000 = V1 (ng). The catecholamine concentration in tissue extract: (A Samp X V1)/A Std = V2 (ng). Total protein in I Samp (µl) injection volume of tissue extract: (P Samp X I Samp ) = V3 (µg). The catecholamine concentration per 1 µg in the injected tissue extract: V2/V3 = V4 (ng/1 µg). The catecholamine concentration per fly head = V5/N = V6 (ng) Injected tissue extract and standard solution was mixed with 5% TCA in a 1:1 ratio. Therefore, the actual catecholamine concentration per fly head (V6/2) = V7 (ng) or (V7 X1000) = V8 (pg). Data analysis Statistical analysis was performed and graphs were prepared using GraphPad Prism 5.0 software. and expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined using a two-tailed unpaired t-test for the data with two groups. For the data with more than two groups, a one-way analysis of variance (ANOVA) followed by the Newman-Keuls Multiple Comparison Test was performed. P-value < 0.05 was considered significant. Result CU rescues “DA neuronal dysfunction” during the health phase but not during the transition phase as determined through the quantification of DAergic neuronal number and the level of Tyrosine hydroxylase synthesis The brain of the adult Drosophila consists of six quantifiable DAergic neuronal clusters in each brain hemisphere. The quantity of DAergic neurons in PAL, PPL1, PPL2, PPM1/2, PPM3, and VUM are 4–5, 11–12, 6/7, 8/9, 5–6 and 3 respectively ( 41 , 51 , 52 ). Utilizing fluorescently labelled secondary antibodies directed against the primary antibody which tags DA synthesizing, tyrosine hydroxylase (TH). Figure 1 A and 2 A, respectively, depicted images of the various experimental groups in Drosophila brain during HP and TP. Results demonstrated that, compared to control group, there is no discernible cluster-wise neuronal number difference in various treatment groups studied during the HP (Fig. 1 B) and TP (Fig. 2 B). Further, total neuronal number in the whole fly brains of different treatment groups did not vary either as compared to control group in both the adult life phases (Fig. 1 C and Fig. 2 C). The “Fluorescence Intensity” (FI) of the DAergic neurons was assessed further to determine if there was any difference/change in the quantity of TH protein production (a secondary antibody that is fluorescently labelled tags the primary antibody anti-TH). Results revealed that the level of TH protein synthesis directly correlates with FI. During HP, in PD brain (Flies fed 10 mM PQ) compared to control, the FI of the DAergic neurons belonging to PAL, PPL1, PPL2, PPM1/2, and PPM3 clusters were significantly decreased by approximately 43%(p < 0.01), 42% (p < 0.01), 41% (p < 0.01), 27% (p < 0.05), and 43% (p < 0.01), respectively (Fig. 1 D). Also, during TP, in PD brain compared to control, FI of the DAergic neurons belonging to PAL, PPL1, PPL2, PPM1/2, and PPM3 clusters were significantly reduced by approximately 30% (p < 0.01), 32% (p < 0.01), 48% (p < 0.001), 29% (p < 0.05) and 25% (p < 0.05) respectively (Fig. 2 D). This indicates that feeding the flies with 10 mM PQ alone significantly reduces the level of TH enzyme (Diminished level of TH synthesis). Characterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining reveals that there is no loss in the number of DAergic neurons upon treatment with PQ during the HP of Drosophila (B,C). However, PQ leads to “neuronal dysfunction” (NDF) as characterized by quantification of DAergic neuronal fluorescence intensity that is proportional to the amount of TH protein and NDF is rescued by CU during the HP (D,E). The significance was drawn by analyzing minimum of three brains followed by two-way ANOVA and Bonferroni post-test for cluster wise analysis. For summative analysis one way ANOVA was performed followed by Tukey post-test. (*p < 0.05; **p < 0.01; ***p < 0.001 compared with PQ treated group; NS- Not significant). The scale bar of all the images in the panel (A) is 20 µm. Represented images are “merged” Z-stacking images; however, the quantification of DAergic neuronal number and fluorescence intensity is performed in 3D Z-stack images (CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM or 1mM, CU1000 µM – CU per se 1mM). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (15%) upon PQ treatment during the HP of Drosophila and CU confers rescue [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM/1mM, CU1000 µM – CU per se 1mM). To access the neuroprotective efficacy of CU at the level of DAergic neurons, CU co-fed groups were assayed compared to PD group. CU intervention rescues cluster-wise depletion of FI during HP (PAL – P < 0.001; PPL1 – P < 0.01, P < 0.001; PPL2 – P < 0.05, P < 0.001; PPM1/2 – P < 0.001; PPM3 – P < 0.01, P < 0.001) (Fig. 1 D) but not during the TP (Fig. 2 D). Characterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining reveals that there is no loss in the number of DAergic neurons upon treatment with PQ at TP of Drosophila (B, C). However, PQ leads to “neuronal dysfunction” (NDF) as characterized by quantification of DAergic neuronal fluorescence intensity that is proportional to the amount of TH protein. However, NDF is not rescued by CU during the TP (D, E). The significance was drawn by analyzing minimum of three brains followed by two-way ANOVA and Bonferroni post-test for cluster wise analysis. For summative analysis one way ANOVA was performed followed by Tukey post-test. (*p < 0.05; **p < 0.01; ***p < 0.001 compared with PQ treated group; NS- Not significant). The scale bar of all the images in the panel (A) is 20 µm. Represented images are “merged” Z-stacking images; however, the quantification of DAergic neuronal number and fluorescence intensity is performed in 3D Z-stack images (CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM or 1mM, CU1000 µM – CU perse 1mM). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (30%) upon PQ treatment at TP and CU fails to rescue this PD phenotype in fly model [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM or 1mM, CU1000 µM – CU per se 1mM). For subsequent investigation, the overall intensity sum of all the DAergic neurons in the fly brain was combined. According to the findings, neurotoxicant exposure led to depletion in the FI (~ 40%; P < 0.001) during HP and (~ 30%; P < 0.001) during TP, suggesting diminished levels of TH protein synthesis. CU intervention rescued the diminished FI significantly during HP (P < 0.001) (Fig. 1 E), suggesting replenishment of TH level/synthesis. However, co-feeding with CU did not replenish the diminished levels of TH protein during the TP (Fig. 2 E). These findings suggest that during HP, but not during TP, CU can reverse the DAergic neurodegeneration/neuronal dysfunction brought upon by PQ exposure. Further, these results substantiate the limitation of nutraceutical CU in alleviating the DAergic neurodegeneration/neuronal dysfunction in TP PD brain. Our insights from immunohistochemistry with fluorescence microscopy were further validated with immunoblotting of the brain-specific TH translate in the ALSS PD model and with CU intervention. We found similar results where neurotoxicant exposure led to diminished TH translate level during both the life stages which was rescued with CU intervention only during HP and not during TP (Fig. 1 F, 2 F). This validates the sensitivity of our fluorescence-based DAergic neuron and TH quantitation method. The findings of the current investigation corroborate the results of the negative geotaxis assay, which showed that CU only corrects mobility defects during the early stages of adult life span but not during the later stages of adult life. PQ diminishes brain DA level and induces alteration in its metabolites (DOPAC and HVA) levels during both health phase and transition phase, whereas CU intervention rescues diminished DA level and altered metabolite level only during health phase but not during transition phase Utilizing the standard and sample chromatogram obtained from the HPLC-ECD unit, the concentration of brain DA and its metabolites were measured to understand DA metabolism (Fig. 3 ). In PD brain of HP (Fig. 4 A), and TP (Fig. 4 B), the DA levels are dramatically reduced by 32% (P < 0.001), and 46% (P < 0.001) respectively. The immediate metabolite of DA, i.e., DOPAC level is also reduced under the PD condition during HP and TP by 18% (P < 0.001) and 40% (P < 0.001) respectively. Decrement of DA and DOPAC levels is also observed in post-mortem brain of PD patients ( 5 ). However, during HP and TP the level of DA depletion is much higher compared to DOPAC (Fig. 4 A,B). This suggests higher DOPAC synthesis compared to DA in the PD condition. HVA is the final product of DA metabolism where in fly through MAO/COMT analogous pathway DA and DOPAC can be degraded to HVA ( 54 , 55 ). Hence, HVA levels were also measured, and was found that in PD brain of the HP, HVA level is increased by 46% (P < 0.001), whereas during TP level of the same is depleted by 29% (P < 0.001). Increment of the HVA level in HP PD brain ( Fig. 4 A) and lesser HVA depletion compared to DA in TP PD brain (Fig. 4 B) suggest that there may also be a higher synthesis HVA in the PD condition. Higher synthesis of DA downstream DOPAC and HVA in the PD condition suggest that these monoamines might have a role in PD onset and progression as they are considered endogenous neurotoxins. CU co-feeding elevated depleted DA level in HP brain significantly (P < 0.001, P < 0.05) (Fig. 4 A). A careful look at the downstream DA metabolite during HP suggests CU intervention further inhibits DOPAC level (P < 0.05, P < 0.001) and gradually normalized enhanced HVA level (NS, P < 0.01) in PD brain in a concentration-dependent manner (Fig. 4 A). Overall, the cumulative insight suggests that CU rescues diminished DA level during HP and simultaneously may prevent DOPAC, HVA synthesis, thereby limiting endogenous toxicity. On the other hand, during TP, it is observed that CU intervention fails to correct depleted DA level (Fig. 4 B) Downstream DA catabolite, DOPAC level which is depleted in PD brain of TP is unaffected by CU intervention, while depleted HVA level is further inhibited (P < 0.01, P < 0.001) in PD brain with CU intervention (Fig. 4 B). During TP, as evident from the observation CU can neither rescue diminished DA level, nor it can fully inhibit endogenous neurotoxicity as only HVA level is downregulated but not DOPAC. Characterization of retention time of standard DOPAC, DA and HVA (A) and brain-specific DA and its metabolites levels (B): Chromatogram of the standard catecholamines gives a particular RT comparing with which the catecholamines in the fly brain sample is analyzed. Quantification of DA levels in fly brain using HPLC. During the HP feeding the flies with PQ alone led to a significant reduction in brain DA and DOPAC levels and increased HVA level (A) Resulting in enhanced DA turnover rate (C). During TP feeding with PQ alone led to a significant reduction in DA level also, however with reduction in the level of DOPAC and HVA (B) which led to prominent DA turnover(D). But co-feeding with CU could significantly alter the diminished DA levels while further inhibiting DOPAC level and preventing HVA level enhancement (A). This results in decrement in DA turnover during HP (C). Further observation suggests that during HP CU per se has no effect on DA pool but inhibits DOPAC level (A). CU intervention during TP fails to rescue diminished DA level and although the intervention further depleted HVA level, it was not enough to prevent DA turnover (B,D). Insights suggest that CU ameliorates DA neuronal degeneration by uplifting DA level and preventing DA breakdown during the earlier stages of adult life phase. Significance was drawn by analyzing the data of minimum three replicates with one-way ANOVA followed by Tukey Post Test for each age group. [*p < 0.05; **p < 0.01; ***p < 0.001; NS: Not significant - compared to PQ treated group], [*C p < 0.05; **C p < 0.01; ***C p < 0.001; NS- Not significant - compared to Control (CTR) group]. Differential regulation of DA oxidative turnover underlies CU-mediated ALSS DAergic neuroprotection: The prominent DA downstream metabolites are DOPAC and HVA. Other than being a natural catabolite of DA, DOPAC and HVA are also known as endogenous neurotoxins as DA oxidation to DOPAC-HVA leads to the production of ROS and peroxides ( 56 , 57 ). The ROS and peroxides in DAergic neurons may be neutralized by the antioxidant defense system. But further, exogenous insults can impair the DA metabolism pathway, enhancing catabolism, and producing more DOPAC-HVA which can promote DAergic neuron degeneration through ROS and peroxide production ( 57 , 58 ). This insight highlights the predisposition of DAergic neurons to degeneration leading to PD phenotype. A study conducted by Stefani et al ., ( 59 ) reports that CSF DOPAC and HVA levels were increased in patients with mild PD symptoms. The increase in DA metabolites is directly correlated to motor impairment. Implication of changes in DA, DOPAC and HVA pools in the PD condition and with CU intervention was further explored to understand DA catabolism and turnover. DA degradation/turnover ratio to its catabolites was calculated with the formula [(DOPAC + HVA)/DA]. It was observed that in PD brain of both life phases that ratio is higher compared to the respective control sample ( Fig. 4 C, 4 D ) . It suggests that the depletion of DA level in the PD condition of both adult life phases may also be due to its degradation to the downstream catabolites i.e., DOPAC and HVA. CU intervention normalizes the DA turnover ratio only during HP but not during TP ( Fig. 4 C, 4 D ) . This observation suggests that oxidative turnover of DA to its catabolites is decreased under CU intervention in PD brain of HP. However, failure of CU intervention to inhibit enhanced DA turnover in PD brain of TP, suggests that the limitation of CU to promote neuroprotection during later adult life stages. These results indicate that CU can rescue “neuronal dysfunction” i.e., diminished TH synthesis during HP but not during TP. Further rescue of TH synthesis results in rescue of diminished DA level during HP but not during TP. CU intervention also rescue altered DOPAC and HVA levels only during HP but not during TP, thereby rescuing the enhanced DA turnover and neurodegeneration. The present study suggests that CU-mediated modulation of perturbations in DA metabolism is restricted to the adult HP and not to the TP. This illustration suggests that the genetic targets and molecular networks of genotropic drug CU-mediated correction process may not be active or expressed at optimum levels during later phases of the adult life. From this result, it can be hypothesized that CU rescues the perturbations in brain DA metabolism in ALSS fashion. This critical observation underlines the limitations of CU as a therapeutic agent in the late-onset NDDs such as PD. Discussion The degeneration of DAergic neurons in the SNpc of the human brain is the characteristic pathological feature associated with PD. PD is a multifactorial disease involving many biochemical pathways, such as oxidative injury/oxidative stress, mitochondrial dysfunction, ER stress, alteration in dopamine catabolism, inactivation of tyrosine hydroxylase (TH), and decrease in the neurotrophic factor BDNF, ultimately resulting in apoptosis of the DAergic neurons in the SNpc ( 5 , 68 ). Fly models of PD also exhibit progressive age-dependent mobility defects, loss of DAergic neurons, and diminished brain DA level in PD condition ( 41 , 60 , 61 ). The distinctive pathological hallmark of PD is the demise of DAergic neurons. Therefore, utilizing fluorescence microscopy, DAergic neurons in the entire fly brain were quantified before characterizing DA "neuronal dysfunction". After DAergic neuronal number quantification, "neuronal dysfunction"( 62 ) if any was deciphered by measuring the FI of fluorescently labelled secondary antibody, which targets the primary anti-TH antibody. The emanating FI can be correlated to the TH protein abundance and synthesis. This was done in order to determine the extent of DAergic neurodegeneration/dysfunction under induced PD conditions and possible neuroprotection with CU intervention. The finding shows that in both the adult health and transition stages of Drosophila , the number of DAergic neurons in the control and PD brains does not differ (Fig. 1 B,C & Fig. 2 B,C). This observation is in line with earlier findings from other studies ( 41 , 62 – 65 ). All these studies make sense in the light of the “dying back” phenomenon which states that neurodegeneration starts from the axonal terminus ( 66 ). The “dying back” of DA neurons further explains the reason behind the failure of time-tested L-DOPA supplementation therapy, where chronic L-DOPA supplementation will lead to its irregular uptake (Due to axonal degeneration) by DAergic neuronal terminals and irregular activation of DA receptors, leading to dyskinesia and toxicity from the plasma L-DOPA ( 67 ). Here, it must be emphasized that there has been a debate in the field of Drosophila neurobiology on the loss of DAergic neuronal cell body (loss in number of DAergic neurons) in fly PD models. The adult-onset loss of DAergic neurons was initially demonstrated by Feany and Bender ( 61 ) in a Drosophila model of PD. Then, several researchers have utilized that model to study and demonstrate the variable degree of DAergic cell death in different DA clusters (68,37,96,93,87,71–74). Auluck and Bonini, ( 75 ); Auluck et al. , ( 69 ); Yang et al. , (76 ) reported a 50% loss of DAergic neurons using the same flies. Similarly, in flies with loss-of-function mutations in PD associated genes like PARKIN , PINK1 , only two to four neurons from a particular DAergic neuronal cluster (PPM1/2 or PPL1) were found to be degenerated ( 77 , 78 , 73 , 79 , 80 ). On the other hand, Pesah et al. , ( 81 ) found no loss of neurons in the PPM1/2 cluster which suggests that in PARKIN mutants, only a specific DAergic cluster might be vulnerable to degeneration. Studies in PINK1 Drosophila model of PD has shown dramatic discrepancies ranging from a discrete loss of two to four neurons in the PPL1 clusters in a null mutant ( 82 ) to a significant decrease in neurons in several DAergic clusters in RNAi knockdown flies ( 83 , 76 ). In addition to the genetic models, toxin-induced PD models viz., PQ based models demonstrated that 5 mM PQ exposure for 12–48 Hrs leads to significant DAergic neuronal loss in PPM and PPL1 cluster ( 84 , 37 , 85 , 86 ). Another independent study by Shukla et al. , ( 87 ) demonstrated that 10 mM, 20 mM PQ exposure for 12 and 24 Hrs leads to cluster-wise selective loss of DAergic neurons which is countered with HSP70 overexpression. Similarly, reduced Aux expression in a fly model shows an alteration in the number of neurons in PPM1/2 cluster which is similar to α-synuclein toxicity ( 70 ). Further, flies with reduced expression of Aux are sensitive to PQ and α-synuclein overexpression suggesting genetic and environmental factors work together in influencing the DAergic neurodegeneration in late HP (33 days old fly) ( 70 ). In PQ induced fly PD model, specific loss of DAergic neurons was found with different concentrations of the toxin ( 84 , 37 , 85 , 86 , 60 , 88 ) or no change in the number of neurons ( 62 ). As such, it can be put forward that there exists a contradiction among researchers in the past and current scenario on the event of DAergic neuronal loss in the Drosophila model of PD. Previously, this matter had been carefully examined in numerous fly models of PD (Both genetic and sporadic), and it has been determined that there is no structural loss of DAergic neurons, rather a decrease in GFP (TH-specific GFP reporter) level/FI, suggesting diminished TH production in DA neurons ( 52 , 51 , 62 ). In the present study flies treated with PQ alone caused a significant reduction in FI in different clusters, which could be significantly rescued upon co-feeding with CU during the HP (Fig. 1 D), but not during the TP (Fig. 2 D). Additionally, an effort was made to evaluate the findings by quantifying the total FI of all the DAergic neurons in the fly brains of various experimental groups (Fig. 1 E & Fig. 2 E). Analyzing these groups separately yields similar results. Reduced levels of FI reflect lower amounts of TH protein (TH signals) because the neuronal cell body's fluorescence is directly correlated with the pace at which the rate-limiting enzyme TH is synthesized. Further, the findings and validity of the novel fluorescent microcopy-based technique developed in our lab was put to test through a well designed traditional immunoblot technique. We found similar trends where the onset of PD diminishes brain TH translate level signifying reduced TH synthesis that could be rescued with CU intervention only during HP but not during TP (Fig. 1 F & Fig. 2 F ). Reduction of TH synthesis but no loss of neuronal cell body is coined as “neuronal dysfunction” which could be the underlying cause of the onset of PD in the current early and late-onset PD model. By quantifying the TH signals, it is possible to quantify incipient neurodegeneration in the PQ-induced fly model and to precisely determine the extent of DAergic neuroprotection through CU intervention. These results validate the observation of Phom et al. , ( 8 ) and suggest that CU rescues the mobility defects and protects DA neuronal dysfunction only during HP of adult Drosophila , but fails to do so during the TP. As the FI of the secondary antibodies is co-related to the level of TH protein synthesis, diminished FI hints at a possible reduction in DA synthesis, which is substantiated by quantifying the brain-specific DA and its metabolites (DOPAC and HVA) through HPLC-ECD. Results demonstrated that neurotoxicant exposure leads to the depletion of DA level in the brain of both phases (Fig. 5 ). The depletion of DA level in the brain of HP is accompanied by the moderate depletion of DOPAC and increment of HVA levels, resulting in increased DA turnover in the PD brain of HP (Fig. 5 ). On the other hand depletion of the DA pool in the TP brain was accompanied by the relatively lesser depletion of DOPAC and HVA, resulting in a moderate increase in DA turnover (Fig. 5 ). In the young mice (6–7 weeks old) extra nigrostriatal DA in nuclear accumbens depletes under PQ-mediated stress. In the same region of the brain, depleted DOPAC level and enhanced HVA level was observed with increased DA turnover when PQ-intoxicated mice were further subjected to psychological stress ( 89 ). Motor and non-motor symptoms of PD patients are further aggravated by the psychological stress resulting in depression. The study by Rudyk et al. , ( 89 ) in a mice model demonstrated that enhanced HVA level with decreased DA, DOPAC level and enhanced DA turnover in some extra nigral brain regions is associated with PD mice having psychological impairment. In the current study in Drosophila with PQ intoxication alone, the HP PD brain shows similar changes in the monoamine pools with increased DA turnover, whereas the TP PD brain shows higher DA turnover (Resulting from higher DOPAC, HVA synthesis) (Fig. 5 ). Further insight is needed to conclude if such change is associated with the onset of psychological disorder in the fly model along with observed PD motor symptoms. In fly models, it was observed that 10 mM or 20 mM PQ exposure on filter paper for 24 hrs reduces DA level and enhances DOPAC level in the brains of adult young flies (2–4 days old) belonging to CS and white eye strains { y W 1118 , Df ( 1 ) w , y } ( 90 , 60 ). The enhanced DOPAC level and lower DA level with the neurotoxicant exposure were postulated to be the enhanced oxidation and degradation DA. In the current study also significant depletion of the DA pool is observed and degradation is manifested in the HP PD brain, owing to a higher HVA pool and a lower depletion of DOPAC compared to DA. The differences in the observation between the current study and Inamdar et al ( 90 )., in regards to DOPAC level modulation in PD brain, although apparent, a closer look suggests otherwise. As observed by Inamdar et al ., a similar concentration (to the current study) of the neurotoxicant exposure enhances DOPAC levels in adult young fly brains (2–4 days old). On the other hand, in the current study in HP PD brain depletion of DA level is higher than that of DOPAC level (Fig. 5 ). This observation in the present study suggests higher DOPAC synthesis from DA oxidation (Therefore lesser DOPAC depletion and higher DA depletion) which corroborates with the hypothesis of Inamdar et al ., ( 90 ). Further, from the current observation it can be postulated that during HP there is a relatively lower level of DOPAC degradation to HVA. Instead, it is possible the HVA is more likely to be synthesized from DA through an alternate route i.e., DA > 3-MT > HVA (Fig. 5 ). Although to a different degree, the synchronous depletion of DA and DOPAC (Fig. 5 ), during the HP PD condition can be explained by the fact that DOPAC is the primary metabolite of DA and as such changes in the DA pool may immediately be reflected on DOPAC pool. In fact, it has been reported that deficiency of DOPAC in the nigrostriatal region and CSF highlights DA deficiency in the central brain and therefore DOPAC pool in CSF is also used as a reliable marker of DA deficiency in the case of human PD ( 91 , 53 ). In the TP brain, it is also observed that the level of DA depletion is at a higher degree compared to DOPAC and HVA (Fig. 5 ). This may, in turn, suggest that there is a higher level of DA oxidative breakdown which may also contribute to PD progression in the aging brain. Although there is no detailed study on DA metabolism in the late-onset fly model of PD, the insight from the current study suggests that PQ-induced sporadic PD condition not only contributes to DA depletion during HP and TP but also enhances DA oxidative breakdown to the downstream catabolites. Owing to the neurotoxic natures of the DA catabolites and the generation of ROS/peroxides due to the catabolic process ( 56 , 57 ) neurodegeneration ensues. During HP DA and DOPAC levels are decreased, while HVA level is increased under PQ-mediated PD condition. The relatively higher DA depletion compared to DOPAC and enhanced HVA suggests, higher DA oxidation in the PD condition in the fly brain. CU intervention rescued diminished DA level and altered DA turnover during HP. In TP PD brain DA, DOPAC and HVA were depleted, although DA depletion was higher compared to DOPAC and HVA. This implied higher DA turnover, but CU intervention failed to rescue diminished DA level and altered DA turnover during TP. Also with natural aging DA, DOPAC and HVA decreased in healthy TP brains. Insight on the CU intervention, suggests that only during HP depleted DA level is rescued with further inhibition of DOPAC level and normalization of enhanced HVA pool, resulting in lesser DA turnover (Fig. 5 ). During TP, however, the DA level is not rescued in the PD brain, DOPAC level remains unaffected and HVA level is further suppressed. As only HVA synthesis is inhibited, but not DOPAC synthesis, CU intervention fails to inhibit the DA turnover in the TP PD brain (Fig. 5 ). CU has been demonstrated to inhibit mitochondrial MAO activity isolated from rat brains ( 92 ). MAO is the primary enzyme necessary for the oxidative turnover of DA to DOPAC and is one of the enzymes necessary for the oxidative turnover of DA to HVA (Fig. 5 ). MAO inhibition is of considerable interest in drug discovery where variants of MAO inhibitors can be used as a potent therapy for neurodegenerative disorders like PD ( 92 ). Although flies do not have the orthologue coding for MAO and COMT enzymes, it is apparent that the fly brain possesses analogous enzymatic pathways for DA catabolism ( 54 ). Therefore, it is possible that during HP, CU intervention not only resuscitates the DA pool in the brain but also inhibits MAO analogous activity, thereby preventing oxidative turnover of DA to its downstream metabolites (Fig. 5 ). Thus, the preservation of DA also prevents ROS generation and promotes neuroprotection during HP, but the same is not possible during TP. Further insights revealed that in healthy aging fly brains, there exists a natural deficiency of DA, DOPAC and HVA pool by 40%, 75% and 66% respectively (Fig. 5 ). This suggests with aging there is an absence and/or deficiency and/or faults in the necessary regulatory players of the catecholamine metabolic pathway. These players may be necessary through which CU might promote neuroprotection at the level of DA metabolism dynamics. Overall, this observation clearly reflects the limitation of CU to promote neuroprotection considering neurochemical pathway modulation during the later phases of adult life (Fig. 5 ). Also, it highlights the possible mode of action of CU in neuroprotection during HP i.e., neuroprotection during HP is brought about by promoting DA synthesis and preventing DA breakdown possibly through MAO inhibition (Fig. 5 ). This is the first report to decipher the neurophysiological and neurochemical aspect of ALSS neuroprotective efficacy of CU (Fig. 6 ). The further study provides insights into the underlying reasons for the neuroprotective efficacy of CU during the health phase and inefficacy during the transition phase. Cartoon summarizes the ALSS- DAergic neuroprotective efficacy of curcumin in Drosophila model of sporadic PD. Paraquat induced fly PD model demonstrate a reduced survivability, mobility defects, neuronal dysfunction and reduced DA and its metabolites, and enhanced levels of DA turnover. Curcumin rescues reduced protein levels of tyrosine hydroxylase, mobility defects, DAergic neuronal dysfunction, DA and its metabolites, and DA turnover rate only during health phase, but fails to rescue during transition phase of adult life during which PD sets in. Curcumin’s life phase specific differential modulation of DA metabolism explains its health phase specific DAergic neuroprotective efficacy. By taking advantage of this knowledge it is possible to develop novel therapeutic strategies and also to modify the existing strategies so that it is feasible to confer protection of DA neurons during later phases of life knowledge of which can be applied to human condition, that will be of great assistance in reducing the burden of disease in PD subjects. The present study explains that curcumin’s ability to modulate perturbed DA metabolism in a PD brain is constrained to the adult health phase. Curcumin-mediated health phase-specific rescue of PD motor deficits underlie rescue of diminished TH synthesis, resulting in rescue of diminished DA level. Further, prevention of DA oxidative turnover by curcumin intervention leads to inhibition of ROS and peroxide generation. In the transition phase similar modulation was not observed with the curcumin intervention which can be attributed to the presence of genetic targets of genotropic nutraceutical curcumin in an adult life phase-specific fashion. Hence, it confers DAergic neuroprotection in the adult health phase but not in the transition phase. The present knowledge relating to life phase-specific modulation of DA metabolism will provide an opportunity to modify the existing therapeutic strategies of PD and will assist to figure-out novel therapeutic strategies to sustain the CU efficacy during late life phases that can be great help to promote the health of aging brain in general and also in different neurodegenerative conditions like PD in particular. Declarations Declaration of competing interest The authors declare that there is no conflict of interest. Data availability statement The raw data data that support the findings of this study are available from the corresponding author upon reasonable request. Acknowledgements This research is supported by the Department of Biotechnology (DBT), India (R&D grant no. BT/405/NE/U-Excel/2013, 11-12-2014). Author Contributions Conceptualization: SCY Data curation: SCY, AD, RC, PM Formal analysis: AD, RC, PM Funding acquisition: SCY Investigation: AD, RC, PM, MA, LP Methodology: SCY, SP,M, PR, BBA Project administration: SCY Resources: SCY Software: AD, RC, PM, MA, LP Supervision: SCY Validation: SCY, AD, RC, MA, PM Visualization: SCY, AD, RC, MA, PM Writing - original draft: AD, RC, PM, MA Writing - review & editing: SCY References Je G, Arora S, Raithatha S, Barrette R, Valizadeh N, Shah U, et al. Epidemiology of Parkinson's disease in rural Gujarat, India. Neuroepidemiol. 2021; 55: 188–195. doi: 10.1159/000515030 Pouchieu C, Piel C, Carles C, Gruber A, Helmer C, Tual S et al. 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Mag. 2016; 12: 116–20. doi: 10.4103/0973-1296.182168 Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformationscirep.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4700590","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":339324299,"identity":"b47b8cf2-e945-4128-af4a-b4a15186648f","order_by":0,"name":"Abhik Das","email":"","orcid":"","institution":"Nagaland University (Central)","correspondingAuthor":false,"prefix":"","firstName":"Abhik","middleName":"","lastName":"Das","suffix":""},{"id":339324300,"identity":"3aa484a8-a7a1-4770-9983-63c37fac1c12","order_by":1,"name":"Rahul Chaurasia","email":"","orcid":"","institution":"Nagaland University 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14:12:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4700590/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4700590/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62402274,"identity":"0159452c-f016-4cdb-a094-8fdade7cce31","added_by":"auto","created_at":"2024-08-13 19:25:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":177487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCU rescues “DA neuronal dysfunction” during the health phase through the quantification of DAergic neuronal number and the level of Tyrosine hydroxylase synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining reveals that there is no loss in the number of DAergic neurons upon treatment with PQ during the HP of \u003cem\u003eDrosophila \u003c/em\u003e(B,C). However, PQ leads to “neuronal dysfunction” (NDF) as characterized by quantification of DAergic neuronal fluorescence intensity that is proportional to the amount of TH protein and NDF is rescued by CU during the HP (D,E). The significance was drawn by analyzing minimum of three brains followed by two-way ANOVA and Bonferroni post-test for cluster wise analysis. For summative analysis one way ANOVA was performed followed by Tukey post-test. (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001 compared with PQ treated group; NS- Not significant). The scale bar of all the images in the panel (A) is 20 µm. Represented images are “merged” Z-stacking images; however, the quantification of DAergic neuronal number and fluorescence intensity is performed in 3D Z-stack images (CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM or 1mM, CU1000 µM – CU \u003cem\u003eper se\u003c/em\u003e 1mM). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (15%) upon PQ treatment during the HP of \u003cem\u003eDrosophila \u003c/em\u003eand CU confers rescue [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM/1mM, CU1000 µM – CU \u003cem\u003eper se\u003c/em\u003e 1mM).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/97a595c1742a876f0ea81d28.jpg"},{"id":62401958,"identity":"799392e2-66cf-4b3f-bf02-15dd810f64ef","added_by":"auto","created_at":"2024-08-13 19:17:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":181025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCUdid notrescue“DA neuronal dysfunction” during the transition phase through the quantification of DAergic neuronal number and the level of Tyrosine hydroxylase synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining reveals that there is no loss in the number of DAergic neurons upon treatment with PQ at TP of \u003cem\u003eDrosophila \u003c/em\u003e(B, C). However, PQ leads to “neuronal dysfunction” (NDF) as characterized by quantification of DAergic neuronal fluorescence intensity that is proportional to the amount of TH protein. However, NDF is not rescued by CU during the TP (D, E). The significance was drawn by analyzing minimum of three brains followed by two-way ANOVA and Bonferroni post-test for cluster wise analysis. For summative analysis one way ANOVA was performed followed by Tukey post-test. (*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001 compared with PQ treated group; NS- Not significant). The scale bar of all the images in the panel (A) is 20 µm. Represented images are “merged” Z-stacking images; however, the quantification of DAergic neuronal number and fluorescence intensity is performed in 3D Z-stack images (CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM or 1mM, CU1000 µM – CU \u003cem\u003eperse\u003c/em\u003e 1mM). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (30%) upon PQ treatment at TP and CU fails to rescue this PD phenotype in fly model [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 µM, R 1000 – PQ along with CU 1000 µM or 1mM, CU1000 µM – CU \u003cem\u003eper se\u003c/em\u003e 1mM).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/9a21efcbf813f08f0d9a764b.jpg"},{"id":62402562,"identity":"a910e8eb-cc9d-4827-be12-46830511fb6a","added_by":"auto","created_at":"2024-08-13 19:33:18","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification and Quantitation of catecholamines in sample\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCharacterization of retention time of standard DOPAC, DA and HVA (A) and brain-specific DA and its metabolites levels (B): Chromatogram of the standard catecholamines gives a particular RT comparing with which the catecholamines in the fly brain sample is analyzed.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/1468469b64c79d138ad9341d.jpg"},{"id":62402273,"identity":"60bf3625-89f6-4c60-8a30-7ece8625a77d","added_by":"auto","created_at":"2024-08-13 19:25:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":122418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePQ diminishes brain DA level and induces alteration in its metabolites (DOPAC and HVA) levels, enhancing DA breakdown during both health phase and transition phase, whereas CU intervention rescues diminished DA level and altered metabolite level, rescuing altered DA breakdown only during health phase but not during transition phase\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQuantification of DA levels in fly brain using HPLC. During the HP feeding the flies with PQ alone led to a significant reduction in brain DA and DOPAC levels and increased HVA level (A) Resulting in enhanced DA turnover rate (C). During TP feeding with PQ alone led to a significant reduction in DA level also, however with reduction in the level of DOPAC and HVA (B) which led to prominent DA turnover(D). But co-feeding with CU could significantly alter the diminished DA levels while further inhibiting DOPAC level and preventing HVA level enhancement (A). This results in decrement in DA turnover during HP (C). Further observation suggests that during HP CU \u003cem\u003eper se\u003c/em\u003e has no effect on DA pool but inhibits DOPAC level (A). CU intervention during TP fails to rescue diminished DA level and although the intervention further depleted HVA level, it was not enough to prevent DA turnover (B,D). Insights suggest that CU ameliorates DA neuronal degeneration by uplifting DA level and preventing DA breakdown during the earlier stages of adult life phase. Significance was drawn by analyzing the data of minimum three replicates with one-way ANOVA followed by Tukey Post Test for each age group. [*p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001; NS: Not significant - compared to PQ treated group], [*C p\u0026lt;0.05; **C p\u0026lt;0.01; ***C p\u0026lt;0.001; NS- Not significant - compared to Control (CTR) group].\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/ef7510f32f5ca0864d53244e.jpg"},{"id":62401961,"identity":"f26e98d8-e91a-40b2-b963-145c7f0ddec3","added_by":"auto","created_at":"2024-08-13 19:17:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":119892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of DA metabolism in PD brain and with CU interventions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring HP DA and DOPAC levels are decreased, while HVA level is increased under PQ-mediated PD condition. The relatively higher DA depletion compared to DOPAC and enhanced HVA suggests, higher DA oxidation in the PD condition in the fly brain. CU intervention rescued diminished DA level and altered DA turnover during HP. In TP PD brain DA, DOPAC and HVA were depleted, although DA depletion was higher compared to DOPAC and HVA. This implied higher DA turnover, but CU intervention failed to rescue diminished DA level and altered DA turnover during TP. Also with natural aging DA, DOPAC and HVA decreased in healthy TP brains.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/aeb1569d8a3c93159e183d2d.jpg"},{"id":62401963,"identity":"0992c9c3-363b-41ca-8c88-df887d584fd6","added_by":"auto","created_at":"2024-08-13 19:17:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":249594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSummary of the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCartoon summarizes the ALSS- DAergic neuroprotective efficacy of curcumin in \u003cem\u003eDrosophila\u003c/em\u003e model of sporadic PD. Paraquat induced fly PD model demonstrate a reduced survivability, mobility defects, neuronal dysfunction and reduced DA and its metabolites, and enhanced levels of DA turnover. Curcumin rescues reduced protein levels of tyrosine hydroxylase, mobility defects, DAergic neuronal dysfunction, DA and its metabolites, and DA turnover rate only during health phase, but fails to rescue during transition phase of adult life during which PD sets in. Curcumin’s life phase specific differential modulation of DA metabolism explains its health phase specific DAergic neuroprotective efficacy. By taking advantage of this knowledge it is possible to develop novel therapeutic strategies and also to modify the existing strategies so that it is feasible to confer protection of DA neurons during later phases of life knowledge of which can be applied to human condition, that will be of great assistance in reducing the burden of disease in PD subjects.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/89e38b12a96d50f78b1e8aea.jpg"},{"id":64432203,"identity":"970ee8bb-45e6-487c-b086-defc10c074ca","added_by":"auto","created_at":"2024-09-13 06:10:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2053651,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/dd30a936-1b41-4267-ab49-5091e0b0a6ef.pdf"},{"id":62402276,"identity":"3d4af1ff-7dda-4235-930a-a08804b10eaa","added_by":"auto","created_at":"2024-08-13 19:25:18","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":204807,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformationscirep.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4700590/v1/034aa31e0350aad7087e86d0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adult Life Phase-Specific Dopaminergic Neuroprotective Efficacy of Curcumin is through Variant Modulation of Brain Dopamine Metabolism: Insights from ALSS Drosophila Model of Parkinson’s Disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a movement disorder prevalent in 1% of the population worldwide. Epidemiological studies have shown a strong correlation between the onset of PD and exposure to environmental neurotoxins such as paraquat (PQ) and rotenone (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The pathogenesis of PD is still yet to be fully elucidated due to the multifactorial nature of the disease. The underlying mechanisms of the dopaminergic (DAergic) neuronal loss are primarily driven by the generation of reactive oxygen species (ROS), decrease in antioxidant enzyme levels, neuroinflammation, mitochondrial dysfunction, and ER stress, leading to a cascade of molecular cross-talks that result in the initiation of neuronal dysfunction (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral animal models of PD have been developed using the herbicide PQ. It has been used in experimental studies focusing on its pathological effects on the brain, heart, lungs, kidneys, liver, and muscle due to systemic toxicity and fatality after acute exposure. The interest in using PQ as a neurotoxin to model PD started since its discovery due to its similarity in terms of its molecular structure and biochemistry with 1-methyl-4-phenylpyridinium (MPP\u003csup\u003e+\u003c/sup\u003e), the active metabolite of MPTP, a neurotoxin that can penetrate the blood brain barrier (BBB) and induce PD-like features in animal models and humans (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe differential expression of multiple genes during the different life stages signifies the process of aging (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). The adult life stages of \u003cem\u003eDrosophila\u003c/em\u003e are categorized into health (no natural death occurs), transition (slight decline in the mortality curve showing 10% death), and senescence stage (steady decline in mortality curve, represented by the window between the end of the transition phase till maximum life span of the fly) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These life stages of \u003cem\u003eDrosophila\u003c/em\u003e, are characterized by different patterns of gene expression (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), like humans (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The transcriptomic analysis of the gene expression profiles in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e has acknowledged 1184 genes with prominent differences in the expression levels between young and old age groups (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). A large number of adult life stage-associated pathways independently influence common and unique complex biological processes of \u003cem\u003eDrosophila\u003c/em\u003e life stages (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). All these studies emphasize the importance and necessity of developing life stage-specific animal models for late-onset NDD such as PD.\u003c/p\u003e \u003cp\u003eStudies in the field of polyphenols and their potential benefits in modern medicine for their positive outcome on human health are becoming very common. Natural products possessing diverse biological activities and drug-like properties are important resources for treating human diseases (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Researchers have suggested the efficacy of natural products by demonstrating their efficacy in modulating biochemical markers, anti-oxidant enzymes and phenotypes associated with the disease in different animal models. Their varied natural role in organisms overlays the basis for their therapeutic prospect in presently non-treatable neurodegenerative disorders including PD. Hence, natural products present in our daily diet that could promote healthy aging are intensively studied.\u003c/p\u003e \u003cp\u003eCurcumin (CU) is an extensively investigated phytochemical with over 20000 PUBMED citations in the last two decades (among these, 303 studies are on PD) and is proven to possess powerful anti-inflammation, anti-viral, anti-oxidant, anti-biotic, anti-depressant, anti-arthritic, wound healing properties, anticancer, and anti-neurodegenerative characteristics (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). It has been employed in 316 clinical trials (116 trials in the USA only) for different human diseases (ClinicalTrial.gov; viewed on 25/11/2023).\u003c/p\u003e \u003cp\u003eThe therapeutic efficacy of CU has been demonstrated in various diseases such as cancer (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), cardiovascular diseases (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), and various neurological disorders such as ALS (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), anxiety (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), depression (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), AD (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), Schizophrenia (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) and PD (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). It has been reported to be a strong forager of a range of ROS, including hydroxyl radicals, superoxide anion radicals, and protection of oxidative damage to kidney cells by suppressing lipid degradation, lipid peroxidation and cellular breakdown (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Randomized clinical trials comprising 631 patients with various diseases have shown the beneficial role of CU/turmeric (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). It is shown to be beneficial in skin diseases such as psoriasis, pruritic skin lesions, radiation dermatitis, vitiligo, etc. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). CU is found to be beneficial in digestive disorders such as indigestion, heartburn, nausea, constipation, diarrhoea, abdominal pain, physical functioning, energy levels and sleep (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). CU is shown to have a beneficial role in several gynaecological disorders (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). CU is also shown to upregulate several genes in an \u003cem\u003ein vitro\u003c/em\u003e human gingival fibroblast wound healing model suggesting its role as a therapeutic agent for gingival ulcers (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Thus, CU is one such potential candidate that can be explored for a therapeutic approach to several human diseases including NDD, like PD.\u003c/p\u003e \u003cp\u003eIt may be noted that the investigation of different phases of life in \u003cem\u003eDrosophila\u003c/em\u003e has shown that each life stage is distinguished by a diverse pattern of gene expression (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). This pattern is comparable to the corresponding life phase in mice, flies and humans. However, several studies have shown the neuroprotective effects of CU by employing young animal models of the adult health phase (\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Therefore, while screening nutraceuticals for their DAergic neuroprotective efficacy in animal models, it is important to follow the adult life stage/phase-specific studies for late-onset NDD such as PD (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe studies concerning CU safety under various experimental conditions are under-reported. Reports suggesting that CU may cause toxicity under specific conditions are shown with the studies in mammalian cell lines on treatment with turmeric in which there is a dose and time-dependent induction of chromosome aberrations (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Another study on the effect of CU on DNA has demonstrated that it induces DNA damage and chromosomal alterations (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). An experiment testing the chelator action of CU to cause iron deficiency \u003cem\u003ein vivo\u003c/em\u003e showed that CU suppressed the production of hepcidin, a peptide that plays an essential role in the regulation and balancing of systemic iron. It causes iron insufficiency, leading to anemia in mice that are fed poor iron diets (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). CU confers negative activity of enzymes such as glutathione-S-transferase and UDP-glucuronosyl transferase apart from hampering the action of drug-metabolizing enzymes such as cytochrome P450 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Such action of CU may result in abnormal plasma flow of certain drugs that may lead to toxic action (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Contradicting the reports that suggest beneficial properties of CU, a study suggested both advantages and disadvantages of CU in alcoholic liver injury. Concentration-dependent CU toxicity was reported in animal models in which CU was found to accelerate liver injury and liver cellular oedema (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a study conducted by our lab to assess the toxicity of CU, it was shown that CU induces dose-dependent lethality in which a concentration of 2.5 mM and above adversely affected the viability of \u003cem\u003eDrosophila\u003c/em\u003e (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Another study showed that a concentration of 1 mg/L aqueous extract of \u003cem\u003eCurcuma longa\u003c/em\u003e induces an adverse effect on the viability of normal SH-SY5Y cells (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHence, the use of CU as a therapeutic agent must be moderated at an optimal dose to avoid its toxic side effects (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Therefore, while developing a therapeutic approach for a disease, it is important to thoroughly validate the toxicity as well as beneficial activity of the drug/phytochemical \u003cem\u003eper se\u003c/em\u003e in the model organism. Taking this important aspect into consideration, our laboratory employed a wide range of CU concentrations and assessed their possible toxicity in the adult life phase-specific \u003cem\u003eDrosophila\u003c/em\u003e model and determined certain sub-lethal concentrations (508). These selected concentrations neither influence viability nor induce mobility defects in \u003cem\u003eDrosophila\u003c/em\u003e and were employed for assessing the neuroprotective efficacy of CU in further studies (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur laboratory has previously demonstrated the adult life stage-specific neuroprotective efficacy of CU in a PQ-mediated \u003cem\u003eDrosophila\u003c/em\u003e model of PD (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Hence, in this study we made an effort to understand the neurophysiological and neurochemical aspects of adult life stage-specific (ALSS) neuroprotective efficacy of CU in the \u003cem\u003eDrosophila\u003c/em\u003e model of sporadic PD. By intervention of CU (500 \u0026micro;M and 1 mM) as was demonstrated in Phom \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), in PD brain.\u003c/p\u003e \u003cp\u003eWe have demonstrated that time-tested nutraceutical CU has limitations to its therapeutic efficacy for late-onset NDD such as PD. The ALSS DAergic neuroprotective efficacy of CU is mediated through the prevention of \u0026ldquo;neuronal dysfunction\u0026rdquo; and differential regulation of brain dopamine (DA) metabolism. This knowledge would help to modulate existing curcumin/nutraceutical mediated therapeutic strategies and assist further in developing efficient healing approaches for late-onset NDD like PD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFly husbandry\u003c/h2\u003e \u003cp\u003eFly was cultured according to the protocol described in Phom \u003cem\u003eet al\u003c/em\u003e. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Oregon K (OK) (procured from National \u003cem\u003eDrosophila\u003c/em\u003e Stock Center, Mysuru University, Mysuru, India) male flies of \u003cem\u003eD. melanogaster\u003c/em\u003e was used in the present study. The flies were raised at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003e0\u003c/sup\u003eC with 12 Hours (Hrs) light and dark cycle in a fly incubator (Percival, USA). The flies were fed with a culture medium composed of sucrose, yeast, agar-agar and propionic acid (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). For collecting the flies, they were mildly anesthetized with a few drops of diethyl ether. Only 25 flies were kept in each vial containing fresh media. The collected flies were transferred to a fresh media vial every third day. 4\u0026ndash;5 day flies were used for further experiments while late health span and transition phase flies were kept transferring routinely for every 3rd day till they reached a specific life stage and then were used for experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eThe required chemicals viz.,Sucrose (SRL, Maharashtra, India,Cat: 84973),Paraquat (PQ; methyl viologen dichloride hydrate,Sigma-Aldrich, St. Louis, MO, United States,Cat: 856177), Curcumin (CU; Sigma-Aldrich, St. Louis, MO, United States,Cat: 1386) and DMSO (Sigma-Aldrich, St. Louis, MO, United States, Cat: D8418) were used for feeding procedures. Standard dopamine (DA; Sigma-Aldrich, St. Louis, MO, United States,Cat: H8502) and its metabolites-3,4-Dihydroxyphenylacetic acid (DOPAC; Sigma-Aldrich, St. Louis, MO, United States, Cat: 11569) and Homovanilic acid (HVA; Sigma-Aldrich, St. Louis, MO, United States,Cat: 69673), Phosphate-buffered Saline (PBS; HiMedia,Maharashtra, India, India, Cat: ML023), Trichloro Acetic Acid (TCA, SRL, Maharashtra, India, Cat: 204842) were used for quantifying DA and metabolites. Paraformaldehyde (Sigma-Aldrich, St. Louis, MO, United States,Cat: I58127), Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States,Cat: T8787), Normal Goat Serum (NGS; Vector Labs, CA, United States,Cat: S1000), VECTASHIELD\u0026reg; mounting medium (Vector Labs, CA, United States,Cat:H1000), Rabbit anti-Tyrosine hydroxylase (anti-TH) polyclonal primary antibody (Millipore, MA, United States, Cat: Ab152) and Goat anti-rabbit IgG H\u0026amp;L(TRITC labelled) polyclonal secondary antibody (Abcam, MA, United States, Cat: Ab6718) were used for immunostaining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTreatment Protocol\u003c/h2\u003e \u003cp\u003eThe fly treatment and CU intervention protocol follows that of Phom \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Briefly, male flies were aged for 4 days (Health phase: HP) and 55 days (Transition phase: TP) feeding in sucrose-agar media. The flies were transferred to freshly prepared media every 3rd day while aging. The flies were then transferred to vials (30mm x100mm) containing disc of Whatman filter paper no. 1 saturated with 275\u0026micro;l of 5% sucrose, 10mM PQ in 5% sucrose, CU (500\u0026micro;M, 1mM) with PQ (10mM) and Curcumin in DMSO. At 24hrs of exposure, flies were frozen at -80˚C. For decapitation of head as required for HPLC, a chilled aluminium tray was positioned on the ice block and flies were placed on it. Using Carl Zeiss stereozoom (Stemi 305) microscope the flies were then dissected separating head from body with a sharp razor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of DAergic neurodegeneration and Tyrosine hydroxylase synthesis in whole fly brain using fluorescence microscopy\u003c/h2\u003e \u003cp\u003eMounting of the whole fly brain for fluorescence microscopy (Carl Zeiss, Axio Imager M2, with \u003cem\u003eZEN 2012 SP2\u003c/em\u003e software, Germany) was done as described in Ayajuddin \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Elaborately, the brains of male Oregon K flies were fixed in 4% paraformaldehyde (PFA) containing 0.5% TritonX (TX)-100, at room temperature for 2 Hrs, and then washed five times afterevery 15 minutes (5 X 15 min) in phosphate-buffered saline (PBS) with 0.1% TX-100 (PBST), at room temperature (RT). Blocking was performed using PBS containing 0.5% TX-100 and 5% normal goat serum (NGS) for 120 minutes at room temperature (RT). Then, primary antibody (anti-TH) incubation was done for the brains with aratio of 1:250 for 72 Hrs at 4\u0026deg; C. The excess primary antibody was washed off from the brains for 5 X 15 minutes with PBST. Brains were then incubated with 1:250 dilution of secondary antibody (TRITC labelled) for 24 Hrs at RT under dark conditions. After thorough washing for 5 X 15 minutes in PBST to remove the excess secondary antibodies, brains were mounted in VECTASHIELD\u0026reg; mounting mediumthen topped with cover glass (Electron Microscopy Sciences,PA, USA), and image acquisition was done on the same day.\u003c/p\u003e \u003cp\u003eThe quantification of DAergic neurons and level of tyrosine hydroxylase (TH) protein synthesis was done as per Ayajuddin \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Briefly, prepared/stained brains were viewed under a fluorescence microscope at a 40x. The image was scanned using a monochromatic camera witha Rhodamine filter using a red dot test for the visibility of neuron(s) and assessing saturation. Then, Z-stack programming with constant intervals was performed. For image processing, on the method column, image subset and maximum intensity projection (MIP) with X\u0026ndash;Y Plane was created.From 3D scan images of Z- stack, PAL, PPL1, PPL2, PPM1/2, PPM3, (PAL- Protocerebral anterior lateral; PPL- Protocerebral posterior lateral; PPM- Protocerebral posterior medial) brain regions were selected. The images were enlarged to see clear neurites, then from graphics appropriate tools \u0026lsquo;draw spline contour\u0026rsquo; were selected and a line was drawn around the neuron creating intensity sum in .xml format. The same process was followed for each neuron located in different clusters. The fly brain with the same orientation was carefully chosen for FI quantification.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein Extraction from\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e \u003cb\u003ebrains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFrom each of the treatments 50 fly heads were homogenized in 160ul of RIPA buffer (50mM Tris HCL, 1% Triton, 0.5% sodium deoxycholate, 150mM NaCl, 0.1% SDS, 2mM EDTA) with protease inhibitor cocktail. Homogenates were then sonicated for 20sec (with pulse of 10sec and amplitude at 30%) using Qsonica sonicators (from OHIO industries). The samples were centrifuged at 13,000rpm for 5min at 4⁰C. Supernatant was re-centrifuged at 13,000rpm for 5min at 4⁰C. Lysates were stored at -80⁰C until quantification was performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProtein quantification\u003c/h2\u003e \u003cp\u003eProtein quantification was performed using the Bio-rad RCDC assay reagents (cat no. 500\u0026thinsp;\u0026minus;\u0026thinsp;0120). Bovine serum albumin at concentration of 1mg/ml (cat no. A-2153, Sigma) was used as the standard and 5ul of the extracted protein lysates were used for quantification of the samples. Absorbance was read at 750nm wavelength.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSDS-polyacrylamide gels were cast using the TGX stain-free fast cast acrylamide kit, 10% from Bio-Rad (cat no 161-0183TA) of 1.5mm thickness.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBio-rad proprietary method for western blotting\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ePrepare resolving gel solution by mixing equal volumes of resolver A and resolver B solution (as described by the manufacturer).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eUse casting stand to stabilize the glass plates during casting.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAdd required volume of TEMED and freshly prepared 10% APS to the combined resolver and mix well. Steadily dispense the solution into the glass plates. Fill the cassette to 1cm below the bottom of the teeth of comb.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ePrepare stacking gel acrylamide solution by combining equal volumes of stacker A and stacker B solution (as described by the manufacturer).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAdd the required volume of TEMED and 10% APS to the stacker solution and mix well. Pipet the solution in the middle of the cassette, filling to the top of short plate. Apply slowly and steadily to prevent mixing with resolving solution. Align and insert the comb in the cassette. Allow the gel to polymerize for 30\u0026ndash;45 min before electrophoresis.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e40ug of each sample lysate were mixed with 20ul of sample buffer (0.5M Tris HCl pH6.8, 10%SDS, glycerol, 0.1% bromophenol blue, β mercaptoethanol) in a total volume of 40ul and denatured for 5min at 95⁰C.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eBio-Rad prestained plus protein dual color standard (161\u0026ndash;0374) was used as the protein ladder marker.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eA 10X stock of running buffer (Tris, Glycine, and SDS) was used for preparing 1X running buffer.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe gel was run at a current of 20mAmp at room temperature using Bio-Rad powerpac basic power system.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eBefore electro blotting, the stain free gels were scanned and activated (2.5min) using the Bio-Rad fluorescent documentation system.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003ePVDF membrane (Bio-Rad 162\u0026ndash;0174) was used after activation by methanol (Merck) before setting up the transfer sandwich.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eTransfer was carried out at a voltage of 90V for the duration of 90min using chilled 1X transfer buffer (Glycine, Tris and methanol) with continuous stirring using magnetic bead. To create a cold temperature condition the transfer tank placed inside a bucket filled with ice.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMembrane blocking and Antibody treatment\u003c/h2\u003e \u003cp\u003ePost transfer the PVDF membrane was scanned using the Bio-Rad fluorescent documentation and incubatedin the blocking buffer of 5% BSA in 1X TBS-T (0.05%) for the duration of 90min at room temperature with gentle rocking. Rabbit polyclonal to Tyrosine Hydroxylase (ab152) was used in the dilution of 1:1000 and the membrane was incubated at 4⁰C for 48hrs. Post primary antibody incubation, the membrane was washed 3X in 1X TBS-T (0.05%) for 15min at room temperature. The secondary antibody Goat anti rabbit HRP (abcam 205718) was used in a dilution of 1:5000 and the membrane was incubated for 24hr at4⁰C. The membrane was washed 5X in 1XTBS-T (0.05%) for 15 min and developed using Clarity western ECL substrate (Bio-Rad 170\u0026ndash;5060). Scanning was performed with Bio-Rad fluorescent documentation system.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData analysis\u003c/b\u003e data analysis was performed using ImageLab 5.2.1 version software\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuantification of brain dopamine and its metabolites using High Performance Liquid Chromatography - Electro chemical detector (HPLC-ECD)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBrain-specific DA and its metabolites were quantified using HPLC-ECD (HPLC-Thermo Scientific, Dionex Ultimate 3000) following the protocol described by Ayajuddin \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Control group and PQ exposed group of flies were immediately frozen following 24 hrs of exposure. To avoid thawing of tissue and degradation of biomolecules, frozen flies were placed on ice tray containing chilled metal surface and 15 fly heads were decapitated quickly with a sharp scalpel. Head tissue homogenate was preparedin 300 \u0026micro;l of chilled PBS. Sonication of the homogenate was performed at 30 percent amplitude for 20 seconds with 5 seconds of interval. Followed by sonication homogenate was centrifuged at 6,000 rpm, 4\u003csup\u003e◦\u003c/sup\u003eC for 10 minutes. After centrifugation, 50 \u0026micro;L of the supernatant was set aside for protein quantification. Rest of the supernatant was combined with 5% TCA (prepared in HPLC grade or enzyme free water) in a 1:1 ratio and kept in ice. Standard DA, DOPAC, HIAAand HVA were prepared in PBS, each having a concentration of 200 ng/ml. The standard solution was mixed with 5% TCA in a 1:1 ratio and kept in ice to prevent catecholamine degradation. For quantification, 50 \u0026micro;l of the tissue sample and 20 \u0026micro;l of the composite standard were loaded into the HPLC. MCM 15 cm X 4.6 mm, 5 \u0026micro; C- 18 packed column (Thermo-Scientific, Waltham, USA, Cat: 70\u0026ndash;0340) was used as the stationary phase for elution of the catecholamines, and MD-TM served as the mobile phase. To detect the catecholamines, the reduction and oxidation potentials within the twocells of primary ECD, were kept at -175 mV and +\u0026thinsp;225 mV respectively. The secondary ECD module acting as third cell also known as Omnicell, was set to +\u0026thinsp;500 mV in order to reduce background noise. Data was gathered at a rate of 5 Hz. Chromatogram analysis was done using Chromeleon\u0026reg;7 from Thermo-Scientific (Waltham, USA). Comparisons were made between sample and standard chromatograms for a catecholamine's retention time. To precisely pinpoint the peaks corresponding to DA, DOPAC, and HVA in the sample, 10 \u0026micro;L of the composite standard was added in sample and run through the HPLC once again. The spiked peaks according to the detection sequence in standard solution were recognized as the catecholamines of interest in sample.\u003c/p\u003e \u003cp\u003eQuantification and normalization of catecholamines is described in Ayajuddin \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn brief, (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Concentration of a catecholamine is: C\u003csub\u003eStd\u003c/sub\u003e (ng/ml), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Area of a catecholamine in the composite standard chromatogram is: A\u003csub\u003eStd,\u003c/sub\u003e and injection volume of the composite standard solution is: I\u003csub\u003eStd\u003c/sub\u003e (\u0026micro;L), (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Area of the catecholamine in the tissue extract chromatogram is: A\u003csub\u003eSamp\u003c/sub\u003e and the injectionvolume of the tissue extract is I\u003csub\u003eSamp\u003c/sub\u003e (\u0026micro;L), (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) Total number of fly heads for protein extraction: N, (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The total protein concentration of the tissue extract is: P\u003csub\u003eSamp\u003c/sub\u003e (\u0026micro;g/\u0026micro;L).\u003c/p\u003e \u003cp\u003eCalculation steps:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe standard catecholamine concentration in I\u003csub\u003eStd\u003c/sub\u003e (\u0026micro;L) injection volume: (C\u003csub\u003eStd\u003c/sub\u003e X I\u003csub\u003eStd\u003c/sub\u003e)/1,000\u0026thinsp;=\u0026thinsp;V1 (ng).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe catecholamine concentration in tissue extract: (A\u003csub\u003eSamp\u003c/sub\u003e X V1)/A\u003csub\u003eStd\u003c/sub\u003e = V2 (ng).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e Total protein in I\u003csub\u003eSamp\u003c/sub\u003e (\u0026micro;l) injection volume of tissue extract: (P\u003csub\u003eSamp\u003c/sub\u003e X I\u003csub\u003eSamp\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;V3 (\u0026micro;g).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe catecholamine concentration per 1 \u0026micro;g in the injected tissue extract: V2/V3\u0026thinsp;=\u0026thinsp;V4 (ng/1 \u0026micro;g).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eThe catecholamine concentration per fly head\u0026thinsp;=\u0026thinsp;V5/N\u0026thinsp;=\u0026thinsp;V6 (ng)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e Injected tissue extract and standard solution was mixed with 5% TCA in a 1:1 ratio. Therefore, the actual catecholamine concentration per fly head (V6/2)\u0026thinsp;=\u0026thinsp;V7 (ng) or (V7 X1000)\u0026thinsp;=\u0026thinsp;V8 (pg).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed and graphs were prepared using GraphPad Prism 5.0 software. and expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical significance was determined using a two-tailed unpaired t-test for the data with two groups. For the data with more than two groups, a one-way analysis of variance (ANOVA) followed by the Newman-Keuls Multiple Comparison Test was performed. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003e \u003cb\u003eCU rescues \u0026ldquo;DA neuronal dysfunction\u0026rdquo; during the health phase but not during the transition phase as determined through the quantification of DAergic neuronal number and the level of Tyrosine hydroxylase synthesis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe brain of the adult \u003cem\u003eDrosophila\u003c/em\u003e consists of six quantifiable DAergic neuronal clusters in each brain hemisphere. The quantity of DAergic neurons in PAL, PPL1, PPL2, PPM1/2, PPM3, and VUM are 4\u0026ndash;5, 11\u0026ndash;12, 6/7, 8/9, 5\u0026ndash;6 and 3 respectively (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Utilizing fluorescently labelled secondary antibodies directed against the primary antibody which tags DA synthesizing, tyrosine hydroxylase (TH). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, respectively, depicted images of the various experimental groups in \u003cem\u003eDrosophila\u003c/em\u003e brain during HP and TP. Results demonstrated that, compared to control group, there is no discernible cluster-wise neuronal number difference in various treatment groups studied during the HP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Further, total neuronal number in the whole fly brains of different treatment groups did not vary either as compared to control group in both the adult life phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC \u003cb\u003eand\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The \u0026ldquo;Fluorescence Intensity\u0026rdquo; (FI) of the DAergic neurons was assessed further to determine if there was any difference/change in the quantity of TH protein production (a secondary antibody that is fluorescently labelled tags the primary antibody anti-TH). Results revealed that the level of TH protein synthesis directly correlates with FI. During HP, in PD brain (Flies fed 10 mM PQ) compared to control, the FI of the DAergic neurons belonging to PAL, PPL1, PPL2, PPM1/2, and PPM3 clusters were significantly decreased by approximately 43%(p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 42% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 41% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 27% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and 43% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Also, during TP, in PD brain compared to control, FI of the DAergic neurons belonging to PAL, PPL1, PPL2, PPM1/2, and PPM3 clusters were significantly reduced by approximately 30% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 32% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 48% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 29% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 25% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). This indicates that feeding the flies with 10 mM PQ alone significantly reduces the level of TH enzyme (Diminished level of TH synthesis).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCharacterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining reveals that there is no loss in the number of DAergic neurons upon treatment with PQ during the HP of \u003cem\u003eDrosophila\u003c/em\u003e (B,C). However, PQ leads to \u0026ldquo;neuronal dysfunction\u0026rdquo; (NDF) as characterized by quantification of DAergic neuronal fluorescence intensity that is proportional to the amount of TH protein and NDF is rescued by CU during the HP (D,E). The significance was drawn by analyzing minimum of three brains followed by two-way ANOVA and Bonferroni post-test for cluster wise analysis. For summative analysis one way ANOVA was performed followed by Tukey post-test. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared with PQ treated group; NS- Not significant). The scale bar of all the images in the panel (A) is 20 \u0026micro;m. Represented images are \u0026ldquo;merged\u0026rdquo; Z-stacking images; however, the quantification of DAergic neuronal number and fluorescence intensity is performed in 3D Z-stack images (CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 \u0026micro;M, R 1000 \u0026ndash; PQ along with CU 1000 \u0026micro;M or 1mM, CU1000 \u0026micro;M \u0026ndash; CU \u003cem\u003eper se\u003c/em\u003e 1mM). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (15%) upon PQ treatment during the HP of \u003cem\u003eDrosophila\u003c/em\u003e and CU confers rescue [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 \u0026micro;M, R 1000 \u0026ndash; PQ along with CU 1000 \u0026micro;M/1mM, CU1000 \u0026micro;M \u0026ndash; CU \u003cem\u003eper se\u003c/em\u003e 1mM).\u003c/p\u003e \u003cp\u003eTo access the neuroprotective efficacy of CU at the level of DAergic neurons, CU co-fed groups were assayed compared to PD group. CU intervention rescues cluster-wise depletion of FI during HP (PAL \u0026ndash; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PPL1 \u0026ndash; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PPL2 \u0026ndash; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PPM1/2 \u0026ndash; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PPM3 \u0026ndash; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) but not during the TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCharacterization of DAergic neurodegeneration in the whole fly brain through anti-TH antibody immunostaining reveals that there is no loss in the number of DAergic neurons upon treatment with PQ at TP of \u003cem\u003eDrosophila\u003c/em\u003e (B, C). However, PQ leads to \u0026ldquo;neuronal dysfunction\u0026rdquo; (NDF) as characterized by quantification of DAergic neuronal fluorescence intensity that is proportional to the amount of TH protein. However, NDF is not rescued by CU during the TP (D, E). The significance was drawn by analyzing minimum of three brains followed by two-way ANOVA and Bonferroni post-test for cluster wise analysis. For summative analysis one way ANOVA was performed followed by Tukey post-test. (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared with PQ treated group; NS- Not significant). The scale bar of all the images in the panel (A) is 20 \u0026micro;m. Represented images are \u0026ldquo;merged\u0026rdquo; Z-stacking images; however, the quantification of DAergic neuronal number and fluorescence intensity is performed in 3D Z-stack images (CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 \u0026micro;M, R 1000 \u0026ndash; PQ along with CU 1000 \u0026micro;M or 1mM, CU1000 \u0026micro;M \u0026ndash; CU \u003cem\u003eperse\u003c/em\u003e 1mM). (F) Stain Free Western Blot analysis shows a reduction of brain TH protein (30%) upon PQ treatment at TP and CU fails to rescue this PD phenotype in fly model [Bio-Rad Stain-Free Western Blotting using total protein normalization method (TPN) (M-protein ladder; CTR- Control; TD- Treated with 10mM PQ; R500- PQ along with CU 500 \u0026micro;M, R 1000 \u0026ndash; PQ along with CU 1000 \u0026micro;M or 1mM, CU1000 \u0026micro;M \u0026ndash; CU \u003cem\u003eper se\u003c/em\u003e 1mM).\u003c/p\u003e \u003cp\u003eFor subsequent investigation, the overall intensity sum of all the DAergic neurons in the fly brain was combined. According to the findings, neurotoxicant exposure led to depletion in the FI (~\u0026thinsp;40%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) during HP and (~\u0026thinsp;30%; P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) during TP, suggesting diminished levels of TH protein synthesis. CU intervention rescued the diminished FI significantly during HP (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), suggesting replenishment of TH level/synthesis. However, co-feeding with CU did not replenish the diminished levels of TH protein during the TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These findings suggest that during HP, but not during TP, CU can reverse the DAergic neurodegeneration/neuronal dysfunction brought upon by PQ exposure. Further, these results substantiate the limitation of nutraceutical CU in alleviating the DAergic neurodegeneration/neuronal dysfunction in TP PD brain. Our insights from immunohistochemistry with fluorescence microscopy were further validated with immunoblotting of the brain-specific TH translate in the ALSS PD model and with CU intervention. We found similar results where neurotoxicant exposure led to diminished TH translate level during both the life stages which was rescued with CU intervention only during HP and not during TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). This validates the sensitivity of our fluorescence-based DAergic neuron and TH quantitation method.\u003c/p\u003e \u003cp\u003eThe findings of the current investigation corroborate the results of the negative geotaxis assay, which showed that CU only corrects mobility defects during the early stages of adult life span but not during the later stages of adult life.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePQ diminishes brain DA level and induces alteration in its metabolites (DOPAC and HVA) levels during both health phase and transition phase, whereas CU intervention rescues diminished DA level and altered metabolite level only during health phase but not during transition phase\u003c/b\u003e \u003c/p\u003e \u003cp\u003eUtilizing the standard and sample chromatogram obtained from the HPLC-ECD unit, the concentration of brain DA and its metabolites were measured to understand DA metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In PD brain of HP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), the DA levels are dramatically reduced by 32% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and 46% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively. The immediate metabolite of DA, i.e., DOPAC level is also reduced under the PD condition during HP and TP by 18% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 40% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) respectively. Decrement of DA and DOPAC levels is also observed in post-mortem brain of PD patients (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, during HP and TP the level of DA depletion is much higher compared to DOPAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA,B). This suggests higher DOPAC synthesis compared to DA in the PD condition. HVA is the final product of DA metabolism where in fly through MAO/COMT analogous pathway DA and DOPAC can be degraded to HVA (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Hence, HVA levels were also measured, and was found that in PD brain of the HP, HVA level is increased by 46% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), whereas during TP level of the same is depleted by 29% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Increment of the HVA level in HP PD brain \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and lesser HVA depletion compared to DA in TP PD brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) suggest that there may also be a higher synthesis HVA in the PD condition. Higher synthesis of DA downstream DOPAC and HVA in the PD condition suggest that these monoamines might have a role in PD onset and progression as they are considered endogenous neurotoxins.\u003c/p\u003e \u003cp\u003eCU co-feeding elevated depleted DA level in HP brain significantly (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). A careful look at the downstream DA metabolite during HP suggests CU intervention further inhibits DOPAC level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and gradually normalized enhanced HVA level (NS, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in PD brain in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Overall, the cumulative insight suggests that CU rescues diminished DA level during HP and simultaneously may prevent DOPAC, HVA synthesis, thereby limiting endogenous toxicity. On the other hand, during TP, it is observed that CU intervention fails to correct depleted DA level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) Downstream DA catabolite, DOPAC level which is depleted in PD brain of TP is unaffected by CU intervention, while depleted HVA level is further inhibited (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in PD brain with CU intervention (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). During TP, as evident from the observation CU can neither rescue diminished DA level, nor it can fully inhibit endogenous neurotoxicity as only HVA level is downregulated but not DOPAC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCharacterization of retention time of standard DOPAC, DA and HVA (A) and brain-specific DA and its metabolites levels (B): Chromatogram of the standard catecholamines gives a particular RT comparing with which the catecholamines in the fly brain sample is analyzed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eQuantification of DA levels in fly brain using HPLC. During the HP feeding the flies with PQ alone led to a significant reduction in brain DA and DOPAC levels and increased HVA level (A) Resulting in enhanced DA turnover rate (C). During TP feeding with PQ alone led to a significant reduction in DA level also, however with reduction in the level of DOPAC and HVA (B) which led to prominent DA turnover(D). But co-feeding with CU could significantly alter the diminished DA levels while further inhibiting DOPAC level and preventing HVA level enhancement (A). This results in decrement in DA turnover during HP (C). Further observation suggests that during HP CU \u003cem\u003eper se\u003c/em\u003e has no effect on DA pool but inhibits DOPAC level (A). CU intervention during TP fails to rescue diminished DA level and although the intervention further depleted HVA level, it was not enough to prevent DA turnover (B,D). Insights suggest that CU ameliorates DA neuronal degeneration by uplifting DA level and preventing DA breakdown during the earlier stages of adult life phase. Significance was drawn by analyzing the data of minimum three replicates with one-way ANOVA followed by Tukey Post Test for each age group. [*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; NS: Not significant - compared to PQ treated group], [*C p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **C p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***C p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; NS- Not significant - compared to Control (CTR) group].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDifferential regulation of DA oxidative turnover underlies CU-mediated ALSS DAergic neuroprotection:\u003c/h2\u003e \u003cp\u003eThe prominent DA downstream metabolites are DOPAC and HVA. Other than being a natural catabolite of DA, DOPAC and HVA are also known as endogenous neurotoxins as DA oxidation to DOPAC-HVA leads to the production of ROS and peroxides (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The ROS and peroxides in DAergic neurons may be neutralized by the antioxidant defense system. But further, exogenous insults can impair the DA metabolism pathway, enhancing catabolism, and producing more DOPAC-HVA which can promote DAergic neuron degeneration through ROS and peroxide production (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). This insight highlights the predisposition of DAergic neurons to degeneration leading to PD phenotype. A study conducted by Stefani \u003cem\u003eet al\u003c/em\u003e., (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e) reports that CSF DOPAC and HVA levels were increased in patients with mild PD symptoms. The increase in DA metabolites is directly correlated to motor impairment. Implication of changes in DA, DOPAC and HVA pools in the PD condition and with CU intervention was further explored to understand DA catabolism and turnover. DA degradation/turnover ratio to its catabolites was calculated with the formula [(DOPAC\u0026thinsp;+\u0026thinsp;HVA)/DA]. It was observed that in PD brain of both life phases that ratio is higher compared to the respective control sample \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. It suggests that the depletion of DA level in the PD condition of both adult life phases may also be due to its degradation to the downstream catabolites i.e., DOPAC and HVA. CU intervention normalizes the DA turnover ratio only during HP but not during TP \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. This observation suggests that oxidative turnover of DA to its catabolites is decreased under CU intervention in PD brain of HP. However, failure of CU intervention to inhibit enhanced DA turnover in PD brain of TP, suggests that the limitation of CU to promote neuroprotection during later adult life stages.\u003c/p\u003e \u003cp\u003eThese results indicate that CU can rescue \u0026ldquo;neuronal dysfunction\u0026rdquo; i.e., diminished TH synthesis during HP but not during TP. Further rescue of TH synthesis results in rescue of diminished DA level during HP but not during TP. CU intervention also rescue altered DOPAC and HVA levels only during HP but not during TP, thereby rescuing the enhanced DA turnover and neurodegeneration. The present study suggests that CU-mediated modulation of perturbations in DA metabolism is restricted to the adult HP and not to the TP. This illustration suggests that the genetic targets and molecular networks of genotropic drug CU-mediated correction process may not be active or expressed at optimum levels during later phases of the adult life. From this result, it can be hypothesized that CU rescues the perturbations in brain DA metabolism in ALSS fashion. This critical observation underlines the limitations of CU as a therapeutic agent in the late-onset NDDs such as PD.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe degeneration of DAergic neurons in the SNpc of the human brain is the characteristic pathological feature associated with PD. PD is a multifactorial disease involving many biochemical pathways, such as oxidative injury/oxidative stress, mitochondrial dysfunction, ER stress, alteration in dopamine catabolism, inactivation of tyrosine hydroxylase (TH), and decrease in the neurotrophic factor BDNF, ultimately resulting in apoptosis of the DAergic neurons in the SNpc (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). Fly models of PD also exhibit progressive age-dependent mobility defects, loss of DAergic neurons, and diminished brain DA level in PD condition (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). The distinctive pathological hallmark of PD is the demise of DAergic neurons. Therefore, utilizing fluorescence microscopy, DAergic neurons in the entire fly brain were quantified before characterizing DA \"neuronal dysfunction\". After DAergic neuronal number quantification, \"neuronal dysfunction\"(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) if any was deciphered by measuring the FI of fluorescently labelled secondary antibody, which targets the primary anti-TH antibody. The emanating FI can be correlated to the TH protein abundance and synthesis. This was done in order to determine the extent of DAergic neurodegeneration/dysfunction under induced PD conditions and possible neuroprotection with CU intervention. The finding shows that in both the adult health and transition stages of \u003cem\u003eDrosophila\u003c/em\u003e, the number of DAergic neurons in the control and PD brains does not differ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB,C \u003cb\u003e\u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB,C). This observation is in line with earlier findings from other studies (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan additionalcitationids=\"CR63 CR64\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). All these studies make sense in the light of the \u0026ldquo;dying back\u0026rdquo; phenomenon which states that neurodegeneration starts from the axonal terminus (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). The \u0026ldquo;dying back\u0026rdquo; of DA neurons further explains the reason behind the failure of time-tested L-DOPA supplementation therapy, where chronic L-DOPA supplementation will lead to its irregular uptake (Due to axonal degeneration) by DAergic neuronal terminals and irregular activation of DA receptors, leading to dyskinesia and toxicity from the plasma L-DOPA (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere, it must be emphasized that there has been a debate in the field of \u003cem\u003eDrosophila\u003c/em\u003e neurobiology on the loss of DAergic neuronal cell body (loss in number of DAergic neurons) in fly PD models. The adult-onset loss of DAergic neurons was initially demonstrated by Feany and Bender (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e) in a \u003cem\u003eDrosophila\u003c/em\u003e model of PD. Then, several researchers have utilized that model to study and demonstrate the variable degree of DAergic cell death in different DA clusters (68,37,96,93,87,71\u0026ndash;74). Auluck and Bonini, (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e); Auluck \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e); Yang \u003cem\u003eet al.\u003c/em\u003e, (76 ) reported a 50% loss of DAergic neurons using the same flies. Similarly, in flies with loss-of-function mutations in PD associated genes like \u003cem\u003ePARKIN\u003c/em\u003e, \u003cem\u003ePINK1\u003c/em\u003e, only two to four neurons from a particular DAergic neuronal cluster (PPM1/2 or PPL1) were found to be degenerated (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). On the other hand, Pesah \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e) found no loss of neurons in the PPM1/2 cluster which suggests that in \u003cem\u003ePARKIN\u003c/em\u003e mutants, only a specific DAergic cluster might be vulnerable to degeneration. Studies in \u003cem\u003ePINK1 Drosophila\u003c/em\u003e model of PD has shown dramatic discrepancies ranging from a discrete loss of two to four neurons in the PPL1 clusters in a null mutant (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e) to a significant decrease in neurons in several DAergic clusters in \u003cem\u003eRNAi\u003c/em\u003e knockdown flies (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). In addition to the genetic models, toxin-induced PD models viz., PQ based models demonstrated that 5 mM PQ exposure for 12\u0026ndash;48 Hrs leads to significant DAergic neuronal loss in PPM and PPL1 cluster (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e). Another independent study by Shukla \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e) demonstrated that 10 mM, 20 mM PQ exposure for 12 and 24 Hrs leads to cluster-wise selective loss of DAergic neurons which is countered with \u003cem\u003eHSP70\u003c/em\u003e overexpression. Similarly, reduced \u003cem\u003eAux\u003c/em\u003e expression in a fly model shows an alteration in the number of neurons in PPM1/2 cluster which is similar to α-synuclein toxicity (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). Further, flies with reduced expression of \u003cem\u003eAux\u003c/em\u003e are sensitive to PQ and α-synuclein overexpression suggesting genetic and environmental factors work together in influencing the DAergic neurodegeneration in late HP (33 days old fly) (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). In PQ induced fly PD model, specific loss of DAergic neurons was found with different concentrations of the toxin (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e) or no change in the number of neurons (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). As such, it can be put forward that there exists a contradiction among researchers in the past and current scenario on the event of DAergic neuronal loss in the \u003cem\u003eDrosophila\u003c/em\u003e model of PD. Previously, this matter had been carefully examined in numerous fly models of PD (Both genetic and sporadic), and it has been determined that there is no structural loss of DAergic neurons, rather a decrease in GFP (TH-specific GFP reporter) level/FI, suggesting diminished TH production in DA neurons (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study flies treated with PQ alone caused a significant reduction in FI in different clusters, which could be significantly rescued upon co-feeding with CU during the HP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), but not during the TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Additionally, an effort was made to evaluate the findings by quantifying the total FI of all the DAergic neurons in the fly brains of various experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE \u003cb\u003e\u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Analyzing these groups separately yields similar results. Reduced levels of FI reflect lower amounts of TH protein (TH signals) because the neuronal cell body's fluorescence is directly correlated with the pace at which the rate-limiting enzyme TH is synthesized. Further, the findings and validity of the novel fluorescent microcopy-based technique developed in our lab was put to test through a well designed traditional immunoblot technique. We found similar trends where the onset of PD diminishes brain TH translate level signifying reduced TH synthesis that could be rescued with CU intervention only during HP but not during TP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF \u003cb\u003e\u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cb\u003e).\u003c/b\u003e Reduction of TH synthesis but no loss of neuronal cell body is coined as \u0026ldquo;neuronal dysfunction\u0026rdquo; which could be the underlying cause of the onset of PD in the current early and late-onset PD model. By quantifying the TH signals, it is possible to quantify incipient neurodegeneration in the PQ-induced fly model and to precisely determine the extent of DAergic neuroprotection through CU intervention. These results validate the observation of Phom \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) and suggest that CU rescues the mobility defects and protects DA neuronal dysfunction only during HP of adult \u003cem\u003eDrosophila\u003c/em\u003e, but fails to do so during the TP. As the FI of the secondary antibodies is co-related to the level of TH protein synthesis, diminished FI hints at a possible reduction in DA synthesis, which is substantiated by quantifying the brain-specific DA and its metabolites (DOPAC and HVA) through HPLC-ECD.\u003c/p\u003e \u003cp\u003eResults demonstrated that neurotoxicant exposure leads to the depletion of DA level in the brain of both phases (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The depletion of DA level in the brain of HP is accompanied by the moderate depletion of DOPAC and increment of HVA levels, resulting in increased DA turnover in the PD brain of HP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). On the other hand depletion of the DA pool in the TP brain was accompanied by the relatively lesser depletion of DOPAC and HVA, resulting in a moderate increase in DA turnover (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the young mice (6\u0026ndash;7 weeks old) extra nigrostriatal DA in nuclear accumbens depletes under PQ-mediated stress. In the same region of the brain, depleted DOPAC level and enhanced HVA level was observed with increased DA turnover when PQ-intoxicated mice were further subjected to psychological stress (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e). Motor and non-motor symptoms of PD patients are further aggravated by the psychological stress resulting in depression. The study by Rudyk \u003cem\u003eet al.\u003c/em\u003e, (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e) in a mice model demonstrated that enhanced HVA level with decreased DA, DOPAC level and enhanced DA turnover in some extra nigral brain regions is associated with PD mice having psychological impairment. In the current study in \u003cem\u003eDrosophila\u003c/em\u003e with PQ intoxication alone, the HP PD brain shows similar changes in the monoamine pools with increased DA turnover, whereas the TP PD brain shows higher DA turnover (Resulting from higher DOPAC, HVA synthesis) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Further insight is needed to conclude if such change is associated with the onset of psychological disorder in the fly model along with observed PD motor symptoms. In fly models, it was observed that 10 mM or 20 mM PQ exposure on filter paper for 24 hrs reduces DA level and enhances DOPAC level in the brains of adult young flies (2\u0026ndash;4 days old) belonging to CS and white eye strains {\u003cem\u003ey W\u003c/em\u003e\u003csup\u003e\u003cem\u003e1118\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eDf\u003c/em\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003cem\u003ew\u003c/em\u003e,\u003cem\u003ey\u003c/em\u003e} (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). The enhanced DOPAC level and lower DA level with the neurotoxicant exposure were postulated to be the enhanced oxidation and degradation DA. In the current study also significant depletion of the DA pool is observed and degradation is manifested in the HP PD brain, owing to a higher HVA pool and a lower depletion of DOPAC compared to DA. The differences in the observation between the current study and Inamdar \u003cem\u003eet al\u003c/em\u003e (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e)., in regards to DOPAC level modulation in PD brain, although apparent, a closer look suggests otherwise. As observed by Inamdar \u003cem\u003eet al\u003c/em\u003e., a similar concentration (to the current study) of the neurotoxicant exposure enhances DOPAC levels in adult young fly brains (2\u0026ndash;4 days old). On the other hand, in the current study in HP PD brain depletion of DA level is higher than that of DOPAC level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This observation in the present study suggests higher DOPAC synthesis from DA oxidation (Therefore lesser DOPAC depletion and higher DA depletion) which corroborates with the hypothesis of Inamdar \u003cem\u003eet al\u003c/em\u003e., (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). Further, from the current observation it can be postulated that during HP there is a relatively lower level of DOPAC degradation to HVA. Instead, it is possible the HVA is more likely to be synthesized from DA through an alternate route i.e., DA\u0026thinsp;\u0026gt;\u0026thinsp;3-MT\u0026thinsp;\u0026gt;\u0026thinsp;HVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Although to a different degree, the synchronous depletion of DA and DOPAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), during the HP PD condition can be explained by the fact that DOPAC is the primary metabolite of DA and as such changes in the DA pool may immediately be reflected on DOPAC pool. In fact, it has been reported that deficiency of DOPAC in the nigrostriatal region and CSF highlights DA deficiency in the central brain and therefore DOPAC pool in CSF is also used as a reliable marker of DA deficiency in the case of human PD (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the TP brain, it is also observed that the level of DA depletion is at a higher degree compared to DOPAC and HVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This may, in turn, suggest that there is a higher level of DA oxidative breakdown which may also contribute to PD progression in the aging brain. Although there is no detailed study on DA metabolism in the late-onset fly model of PD, the insight from the current study suggests that PQ-induced sporadic PD condition not only contributes to DA depletion during HP and TP but also enhances DA oxidative breakdown to the downstream catabolites. Owing to the neurotoxic natures of the DA catabolites and the generation of ROS/peroxides due to the catabolic process (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) neurodegeneration ensues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDuring HP DA and DOPAC levels are decreased, while HVA level is increased under PQ-mediated PD condition. The relatively higher DA depletion compared to DOPAC and enhanced HVA suggests, higher DA oxidation in the PD condition in the fly brain. CU intervention rescued diminished DA level and altered DA turnover during HP. In TP PD brain DA, DOPAC and HVA were depleted, although DA depletion was higher compared to DOPAC and HVA. This implied higher DA turnover, but CU intervention failed to rescue diminished DA level and altered DA turnover during TP. Also with natural aging DA, DOPAC and HVA decreased in healthy TP brains.\u003c/p\u003e \u003cp\u003eInsight on the CU intervention, suggests that only during HP depleted DA level is rescued with further inhibition of DOPAC level and normalization of enhanced HVA pool, resulting in lesser DA turnover (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). During TP, however, the DA level is not rescued in the PD brain, DOPAC level remains unaffected and HVA level is further suppressed. As only HVA synthesis is inhibited, but not DOPAC synthesis, CU intervention fails to inhibit the DA turnover in the TP PD brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). CU has been demonstrated to inhibit mitochondrial MAO activity isolated from rat brains (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). MAO is the primary enzyme necessary for the oxidative turnover of DA to DOPAC and is one of the enzymes necessary for the oxidative turnover of DA to HVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). MAO inhibition is of considerable interest in drug discovery where variants of MAO inhibitors can be used as a potent therapy for neurodegenerative disorders like PD (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). Although flies do not have the orthologue coding for MAO and COMT enzymes, it is apparent that the fly brain possesses analogous enzymatic pathways for DA catabolism (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). Therefore, it is possible that during HP, CU intervention not only resuscitates the DA pool in the brain but also inhibits MAO analogous activity, thereby preventing oxidative turnover of DA to its downstream metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Thus, the preservation of DA also prevents ROS generation and promotes neuroprotection during HP, but the same is not possible during TP. Further insights revealed that in healthy aging fly brains, there exists a natural deficiency of DA, DOPAC and HVA pool by 40%, 75% and 66% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This suggests with aging there is an absence and/or deficiency and/or faults in the necessary regulatory players of the catecholamine metabolic pathway. These players may be necessary through which CU might promote neuroprotection at the level of DA metabolism dynamics. Overall, this observation clearly reflects the limitation of CU to promote neuroprotection considering neurochemical pathway modulation during the later phases of adult life (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Also, it highlights the possible mode of action of CU in neuroprotection during HP i.e., neuroprotection during HP is brought about by promoting DA synthesis and preventing DA breakdown possibly through MAO inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis is the first report to decipher the neurophysiological and neurochemical aspect of ALSS neuroprotective efficacy of CU (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The further study provides insights into the underlying reasons for the neuroprotective efficacy of CU during the health phase and inefficacy during the transition phase.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCartoon summarizes the ALSS- DAergic neuroprotective efficacy of curcumin in \u003cem\u003eDrosophila\u003c/em\u003e model of sporadic PD. Paraquat induced fly PD model demonstrate a reduced survivability, mobility defects, neuronal dysfunction and reduced DA and its metabolites, and enhanced levels of DA turnover. Curcumin rescues reduced protein levels of tyrosine hydroxylase, mobility defects, DAergic neuronal dysfunction, DA and its metabolites, and DA turnover rate only during health phase, but fails to rescue during transition phase of adult life during which PD sets in. Curcumin\u0026rsquo;s life phase specific differential modulation of DA metabolism explains its health phase specific DAergic neuroprotective efficacy. By taking advantage of this knowledge it is possible to develop novel therapeutic strategies and also to modify the existing strategies so that it is feasible to confer protection of DA neurons during later phases of life knowledge of which can be applied to human condition, that will be of great assistance in reducing the burden of disease in PD subjects.\u003c/p\u003e \u003cp\u003eThe present study explains that curcumin\u0026rsquo;s ability to modulate perturbed DA metabolism in a PD brain is constrained to the adult health phase. Curcumin-mediated health phase-specific rescue of PD motor deficits underlie rescue of diminished TH synthesis, resulting in rescue of diminished DA level. Further, prevention of DA oxidative turnover by curcumin intervention leads to inhibition of ROS and peroxide generation. In the transition phase similar modulation was not observed with the curcumin intervention which can be attributed to the presence of genetic targets of genotropic nutraceutical curcumin in an adult life phase-specific fashion. Hence, it confers DAergic neuroprotection in the adult health phase but not in the transition phase. The present knowledge relating to life phase-specific modulation of DA metabolism will provide an opportunity to modify the existing therapeutic strategies of PD and will assist to figure-out novel therapeutic strategies to sustain the CU efficacy during late life phases that can be great help to promote the health of aging brain in general and also in different neurodegenerative conditions like PD in particular.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is supported by the Department of Biotechnology (DBT), India (R\u0026amp;D grant no. BT/405/NE/U-Excel/2013, 11-12-2014). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u003c/strong\u003eSCY\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u003c/strong\u003eSCY, AD, RC, PM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormal analysis:\u003c/strong\u003eAD, RC, PM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition:\u003c/strong\u003eSCY\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003eAD, RC, PM, MA, LP\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003eSCY, SP,M, PR, BBA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003eSCY\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003eSCY\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoftware:\u003c/strong\u003eAD, RC, PM, MA, LP\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003eSCY\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation:\u003c/strong\u003eSCY, AD, RC, MA, PM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization:\u0026nbsp;\u003c/strong\u003eSCY, AD, RC, MA, PM\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting - original draft:\u003c/strong\u003eAD, RC, PM, MA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting - review \u0026amp; editing:\u003c/strong\u003eSCY\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJe G, Arora S, Raithatha S, Barrette R, Valizadeh N, Shah U, et al. 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Mag. 2016; 12: 116\u0026ndash;20. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4103/0973-1296.182168\u003c/span\u003e\u003cspan address=\"10.4103/0973-1296.182168\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"Curcumin, Dopamine, Drosophila, Health phase, Parkinson’s disease, Paraquat, Transition stage","lastPublishedDoi":"10.21203/rs.3.rs-4700590/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4700590/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpidemiological studies suggest a strong link between exposure to environmental toxins and onset of Parkinson\u0026rsquo;s disease (PD). Our laboratory has developed an adult life stage-specific (ALSS) \u003cem\u003eDrosophila\u003c/em\u003e model of sporadic PD which is critical to screen small molecules and identify molecular targets of dopaminergic (DAergic) neuroprotection for late-onset neurodegenerative diseases (NDD) such as PD. Nutraceutical curcumin (CU) has been a time-tested ingredient in the Asian kitchen, traditional medicine and has been employed in 450 clinical trials. Exposure to Paraquat (PQ) induces mobility defects in the health stage (point of time when there is no natural diseases) and transition stage (time period wherein about 10% deaths occur) of adult \u003cem\u003eDrosophila;\u003c/em\u003e whereas CU ameliorates the deficits only during early health span but fails during late health and transition phases. Probing the whole fly brain using anti-tyrosine hydroxylase (anti-TH) antibodies, for PQ-mediated dopamine (DA) neurodegeneration illustrates that it does not cause loss of DA neurons \u003cem\u003eper se\u003c/em\u003e. However, it leads to DA \u0026ldquo;neuronal dysfunction\u0026rdquo; (diminished levels of rate-limiting enzyme in dopamine synthesis- TH) and CU rescues the neuronal dysfunction only during the early health span but fails to mitigate the DA neuronal pathology during the transition phase of adult life. Genotropic nutraceutical CU replenishes the diminished levels of brain-specific DA and its metabolites DOPAC and HVA during the adult health phase (HP) and fails to do so in the adult transition phase (TP), suggesting its life phase-specific dopaminergic neuroprotective efficacy is mediated through differential modulation of perturbations in brain dopamine metabolism. The present study suggests the limitation of CU as a therapeutic strategy for PD and emphasizes the necessity and importance of screening putative neuroprotective small molecules for late onset NDD such as PD in life phase matched animal models during which the disease sets in.\u003c/p\u003e","manuscriptTitle":"Adult Life Phase-Specific Dopaminergic Neuroprotective Efficacy of Curcumin is through Variant Modulation of Brain Dopamine Metabolism: Insights from ALSS Drosophila Model of Parkinson’s Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-13 19:17:13","doi":"10.21203/rs.3.rs-4700590/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":"6b2948f4-290d-4b41-936e-a3c828c4a356","owner":[],"postedDate":"August 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35947202,"name":"Biological sciences/Biochemistry"},{"id":35947203,"name":"Biological sciences/Cell biology"},{"id":35947204,"name":"Biological sciences/Developmental biology"},{"id":35947205,"name":"Biological sciences/Neuroscience"},{"id":35947206,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2024-09-13T06:02:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-13 19:17:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4700590","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4700590","identity":"rs-4700590","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}