Age specific neuroprotection of curcumin is through differential modulation of brain dopamine metabolism: Insights from Drosophila model of Parkinson’s disease 

Age specific neuroprotection of curcumin is through differential modulation of brain dopamine metabolism: Insights from 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 Age specific neuroprotection of curcumin is through differential modulation of brain dopamine metabolism: Insights from Drosophila model of Parkinson’s disease Mohamad Ayajuddin, Abhik Das, Sarat Yenisetti This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4645640/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 linkage between exposure to environmental toxins and onset of Parkinson’s disease (PD). Rotenone is a widely used pesticide and known inhibitor of mitochondrial complex I, that has been shown to induce Parkinsonian phenotypes in various animal models. Our laboratory has developed a rotenone mediated ALSS Drosophila model of PD which is critical to screen small molecules and identify molecular targets of dopaminergic neuroprotection for late-onset neurodegenerative diseases such as PD. Using negative geotaxis assay, qualitative and quantitative analysis of dopaminergic neurons by fluorescence microscopy and further quantifying the levels of dopamine and its metabolites by HPLC, we have assessed the neurodegeneration under PD induced conditions and neuroprotection by employing curcumin in Drosophila model of PD. Exposure to rotenone induces mobility defects in health and transition phase of adult Drosophila; whereas curcumin ameliorates the deficits only during early health phase but fail during late health and transition phases. Probing the whole fly brain using anti-tyrosine hydroxylase antibodies, for rotenone mediated dopamine neurodegeneration illustrates that it does not cause loss of dopaminergic neurons per se . However, it leads to dopaminergic “neuronal dysfunction” (diminished levels of rate limiting enzyme of dopamine synthesis) and curcumin rescues the neuronal dysfunction only during the early health phase but fails to mitigate the dopamine neuronal pathology during the transition phase of adult life. Genotropic nutraceutical curcumin replenishes the diminished levels of brain specific dopamine and its metabolites DOPAC and HVA during adult early health phase and fails to do so in adult transition phase, suggesting that the life phase-specific dopaminergic neuroprotective efficacy is mediated through differential modulation of perturbations in brain dopamine metabolism. Present study suggests the limitation of curcumin as a therapeutic agent for PD and emphasizes the necessity of screening putative neuroprotective small molecules for late onset neurodegenerative diseases such as PD in life phase matched animal models during which the disease sets in. Biological sciences/Cell biology Biological sciences/Neuroscience Biological sciences/Zoology Curcumin Dopamine Drosophila Health phase Parkinson’s disease Rotenone Transition stage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Parkinson’s disease (PD) is a movement disorder suffering among 1% of the population worldwide. Epidemiological and animal model studies suggest a strong correlation between the onset of PD and exposure to pesticides and other environmental toxins [ 1 – 3 ]. Several animal models of PD have been developed using the herbicide rotenone as a neurotoxicant [ 4 – 6 ]. Rotenone can cross the blood brain barrier, impair the oxidative phosphorylation of the electron transport system (ETC) of mitochondria generating reactive oxygen species (ROS) and further inducing apoptosis and cytotoxicity [ 7 , 8 ] leading to the pathological characteristics of PD including dopaminergic (DAergic) neurodegeneration, depletion of dopamine levels in the brain, and mitochondrial dysfunction [ 5 , 9 ]. The differential expression of multiple genes during the different life stages signifies the process of aging [ 10 , 11 ]. The adult life stages of Drosophila is 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) [ 12 ]. These life stages of model organisms such as mice and fly, are characterized by different patterns of gene expression [ 11 , 13 ], parallel with the equivalent life stages of humans. For instance, 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 [ 10 ]. A large number of stage-associated pathways independently influence a common and unique complex process of the life stages of Drosophila [ 14 ]. All these studies emphasize the importance and necessity of developing life stage-specific animal models for late-onset NDDs such as PD. Hence, our laboratory made a comprehensive effort and recently developed a neurotoxin rotenone (which inhibits mitochondrial complex I of ETC) induced adult life stage-specific (ALSS) Drosophila model of sporadic PD [ 5 ]. This novel model is a good tool to understand the progression of pathophysiology and to screen potential therapeutic compounds/nutraceuticals and identify their molecular targets of activity, knowledge of which will assist in developing novel therapeutic strategies for 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 [ 15 ]. Researchers have suggested the efficacy of natural products by demonstrating their efficacy to modulate biochemical markers, anti-oxidant enzymes and phenotypes associated with the disease in different animal models. Their varied natural role in organisms overlay 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 (CUR) is an extensively investigated phytochemical with 23,314 PUBMED articles, among which 316 articles are on PD (as on 15/04/2024). It possess powerful anti-inflammatory and anti-oxidant properties thereby exhibits significant neuroprotective properties by modulating neuro-inflammatory pathways, scavenging reactive oxygen species, and inhibiting the production of pro-inflammatory cytokines [ 16 ]. The therapeutic efficacy of CUR has been demonstrated in various diseases including PD [ 17 , 18 ]. Randomized clinical trials comprising 631 patients with various diseases have shown the beneficial role of CUR/turmeric [ 19 ]. Thus, CUR is one such potential candidate that can be explored for a therapeutic approach to several human diseases including NDDs like PD. However, the investigation of different phases of life in Drosophila has shown that each life stage is distinguished by a diverse pattern of gene expression [ 11 , 20 ]. This pattern is comparable to the corresponding life phase in mice, fly and humans. Nevertheless, several studies have shown the neuroprotective effects of CUR by employing young animal models of the adult health phase [ 6 , 21 , 22 ]. Therefore, while screening nutraceuticals for their DA-ergic neuroprotective efficacy in animal models, it is important to follow the adult life stage/phase-specific studies for late-onset NDD such as PD [ 5 , 23 ]. Our laboratory has previously demonstrated the ALSS neuroprotective efficacy of CUR in paraquat-mediated Drosophila model of PD and also dose-dependent lethality of CUR per se in Drosophila [ 23 ]. Further, our laboratory recently developed adult health and transition stage specific PD model by inhibiting brain mitochondrial complex I using environmental neurotoxicant rotenone [ 5 ]. This model is closer to human PD condition and in order to validate the model we employed curcumin and demonstrated that its efficacy is ALSS. Here, we made an effort to understand the neuroprotective efficacy of CUR in ROT-mediated health and transition phase-specific Drosophila model of PD by employing four different concentrations of CUR (100 µM, 250 µM, 500 µM and 1000 µM) in both the pre- and co-feeding regimen through assessing mobility phenotypes and further by characterizing the DA-ergic neurodegeneration/protection and modulation of DA metabolism and possible rescue/no rescue under induced PD conditions. For first time, we have demonstrated that time tested nutraceutical CUR has limitations to its therapeutic efficacy for late-onset NDDs such as PD and ALSS dopaminergic neuroprotective efficacy of CUR is mediated through differential regulation of brain dopamine metabolism and 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. 2. Materials and Methods 2.1 Fly husbandry Fly was cultured according to the protocol described in Ayajuddin et al. [ 5 ]. Oregon K (OK) (procured from National Drosophila Stock Center, Mysuru University, Mysuru, India) male flies of D. melanogaster were used in the present study. The flies were raised at 22 ± 1 0 C with 12 Hour (Hr) 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 (Phom et al., 2014). For collecting the flies, they were mildly anaesthetized 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. Four-five days old flies were used for further experiment while late health span and transition phase flies were kept transferring routinely every 3rd day till they reached a specific life stage and then were used for experiments. 2.2 Chemicals The required chemicals viz., Rotenone (Sigma, Cat: R8875), Curcumin (Sigma, Cat: 1386) and DMSO (Sigma, Cat: D8418) were used for feeding procedures. Standard dopamine (DA; Sigma-Aldrich, Cat: H8502) and its metabolites- 3,4-Dihydroxyphenylacetic acid (DOPAC; Sigma-Aldrich, Cat: 11569) and Homovanilic acid (HVA; Sigma-Aldrich, Cat: 69673) were used for quantifying DA and metabolites. Paraformaldehyde (Sigma, Cat: I58127), Triton X-100 (Sigma, Cat: T8787), Normal Goat Serum (NGS; Vector Lab, Cat: S1000), VECTASHIELD mounting medium (Vector Labs, CA, USA, Cat: H1000), Rabbit anti-Tyrosine hydroxylase (anti-TH) polyclonal primary antibody (Millipore, MA, USA, Cat: Ab152) and Goat anti-rabbit IgG H&L (TRITC labelled) polyclonal secondary antibody (Abcam, MA, USA, Cat: Ab6718) were used for immunostaining. 2.3 Negative geotaxis assay The upward mobility of the flies, also called negative geotaxis assay, was assessed as described in Ayajuddin et al. [ 5 ]. In short, a single fly aspirated out of the vial was released into a plastic tube and acclimatized for 2 minutes. After gently tapping the fly to the bottom of the tube, the distance it could climb in 12 seconds was recorded. The same fly was given three chances and a minimum of 12 flies were noted for each concentration. 2.4 Curcumin toxicity assay Four to five days and 50 days old Drosophila males were fed on eight different concentrations of curcumin (25 µM, 50 µM, 100 µM, 250 µM, 500 µM, 1 mM, 1.5mM and 2.5 mM). Mortality was recorded every 24 Hr for 10 days. The concentrations at which no mortality was observed during this period were selected for further study to understand the protective efficacy of this molecule. Control flies were fed on 5% sucrose. 2.5 Curcumin pre- and co-feeding regimens For understanding the efficacy of CUR in the Drosophila model of PD, two treatment regimens were employed i.e., Co-feeding regime and Pre-feeding regime. The concentrations of ROT used for the experiments were 500 µM, 25 µM and 10 µM for early health phase, late health phase and transition phase flies respectively. All these concentrations were selected as described in Ayajuddin et al. (2022). CUR concentrations were selected as described in the above section. Figure 1 : Co-feeding and pre-feeding regimen using Drosophila : (A) Co-feeding : Flies belonging to different life phases (5 days: Early health span; 30 days: Late health span; and 50 days: transition phase) were fed with ROT alone or ROT and CUR for 5 days in case of early health phase and 2 days in case of late health phase and transition phase flies. Control fly remained in 5% sucrose only while the CUR (500 µM) per se group was fed with CUR only. The negative geotaxis assay (NGA) was performed on the 4th and 5th day of exposure to ROT in the case of early health phase fly and on 1st and 2nd day of exposure to ROT in case of the late health phase and transition phase flies respectively. (B) Pre-feeding regimen : Flies of different life stages (Early health phase; late health phase; and transition phase) were pre-fed with CUR for 3 days. Control and the group to be treated with ROT remain in 5% sucrose during this period. The fly was then exposed to ROT for 5 days in case of the early health phase and 2 days in case of the late health phase and transition phase flies. Control and CUR (500 µM) per se group remain in 5% sucrose. NGA was performed on 4th and 5th day after exposure to ROT in case of the early health phase fly and on 1st and 2nd day of exposure to ROT in case of the late health phase and transition phase flies. (A) Co-feeding regime : In the co-feeding regimen, the fly was fed with ROT alone for ROT treatment group and a combination of ROT along with CUR (100 µM, 250 µM, 500 µM and 1000 µM) for the co-feeding group while the control fly remain in 5% sucrose only. (B) Pre-feeding regime : In the pre-feeding regimen, fly was fed with CUR (100 µM, 250 µM, 500 µM and 1000 µM) for 2, 3 and 5 days and then switched to ROT. For the ROT treatment group, fly was fed with 5% sucrose for 2, 3, 4 and 5 days and then transferred to ROT while the control fly was fed with 5% sucrose only. Climbing ability was assessed on the 4th and 5th day of feeding with ROT for the early health phase; while it was on 1st and 2nd day for the late health span and transition phase flies ( Fig. 1 ). 2.6 Quantification of dopaminergic neurodegeneration and tyrosine hydroxylase synthesis in whole fly brain using fluorescence microscopy: Quantification of dopaminergic neurons and fluorescence intensity (FI) was done according to Chaurasia et al. [ 24 ]. Briefly, Briefly, the male OK flies were fixed in 4% paraformaldehyde (PFA) containing 0.5% Triton (TX)-100, at room temperature for 2 Hr, and then washed 5 times after every 15 minutes in phosphate-buffered saline with 0.1% TX-100 (PBST), at room temperature (RT). Blocking was done using 0.5% TX-100 and 5% NGS for 120 minutes at RT. Then, the brains were incubated in primary antibody (anti-TH) diluted in 1:250 ratio for 72 Hr at 4°C. The excess primary antibody was removed by washing the brains for 5X15 minutes in PBST. Brains were then incubated with a 1:250 dilution of secondary antibody (TRITC labelled) for 24 Hr at room temperature under dark conditions. After thorough washing for 5X15 minutes in PBST, brains were mounted in VECTASHIELD mounting medium and image acquisition was done on the same day. The idea to quantify the FI of fluorescently labelled secondary antibodies (using Carl Zeiss, ZEN 2012 SP2 software) was adapted from the protocol of Navarro et al. [ 25 ], where they quantified the FI of GFP reporter. In brief, prepared/stained brains were viewed under a fluorescence microscope. Rhodamine filter was used for scanning the image. For image acquisition at 40x, red dot test for visibility of neurons was done for all the brains. Z-stack programming with constant intervals was performed. For image processing, on method column, image subset and maximum intensity projection (MIP) with X-Y Plane were created. From 3D 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 and a line was drawn around the neuron using a draw spline contour from graphics tools and the intensity sum was created in .xml format. The procedure was repeated for all the neurons in different clusters. Care was taken to select the fly brains with the same orientation. 2.7 Quantification of brain dopamine and its metabolites using HPLC-ECD Brain-specific dopamine and its metabolites were quantified using High-Performance Liquid Chromatography (HPLC-Thermo Scientific, Dionex Ultimate 3000) equiped with an electrochemical detector (ECD) as described in Ayajuddin et al. [ 5 ]. Briefly, the brains were homogenized in ice-chilled PBS. The supernatant collected after centrifugation was mixed with 5% TCA in a ratio of 1:1. 50 µL of the supernatant was kept aside for protein quantification before mixing with TCA. 20 µL of standard DA, HVA, DOPAC and 50 µL of the sample were loaded onto HPLC for quantification. MD-TM from Thermo Scientific (Cat: 701332) was used as the mobile phase and MCM 15 cm X 4.6 mm, 5 µ C-18 packed column (Thermo Scientific, Cat: 70–0340) was used as a stationary phase for elution of the monoamines. Inside the primary ECD containing two cells, detection of the monoamines was done at a range of -175 mV to + 225 mV as reduction and oxidation potential respectively. The third cell which is part of the Omnicell (secondary ECD module) was set at + 500 mV to reduce background noise. The data collection rate was set at 5 Hz. Chromatograms were analyzed using Chromaleon® 7, Thermo Scientific, United States 2.8 Data analysis Statistical analysis was performed 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, one-way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test was performed. P-value < 0.05 was considered significant. 3. Result 3.1 CUR toxicity studies: Drosophila is susceptible to CUR in a time-dose-dependent manner In order to understand the susceptibility of Drosophila to CUR, male OK flies of early health and transition phase were fed with different concentrations of CUR (25 µM, 50 µM, 100 µM, 250 µM, 500 µM, 1 mM, 1.5 mM and 2.5 mM) while the control flies remain on 5% sucrose only. The survivorship of flies was observed and recorded every 24 Hr till all the flies were dead. A total of 100 flies were used for each concentration. Figure 2 : Dose- and time-dependent mortality of OK male fly exposed to CUR during different phases of adult life : Mortality pattern among adult male flies of early health phase ( A ) and transition phase ( B ), exposed to eight different concentrations of CUR (25 µM, 50 µM, 100 µM, 250 µM, 500 µM, 1 mM, 1.5 mM and 2.5 mM) showed concentration-dependent lethality. Mortality data were collected every 24 Hr for each group till all the flies were dead. A comparison of survival curves showed a significant difference in response among different tested concentrations (log-rank [Mantel–Cox] test, p < 0.0001). CUR exerts concentration-dependent lethality on both the life stages of the fly. The survivability of the flies in the concentrations of 25 µM, 50 µM, 100 µM and 250 µM were very close to the control group. The concentrations of CUR ranging from 25 µM to 1 mM showed no observable toxicity while concentrations of 1.5 mM and 2.5mM affect the viability of the transition phase fly showing that higher concentrations of CUR could be harmful to the fly. The fly in the lowest concentration of 25 µM could survive for 55 days (maximum life span) while it could survive for 30 days in the highest concentration of 2.5 mM in the case of the early health phase fly. In the case of the transition phase fly, it could survive for only 30 days in the lowest concentration of 25 µM and 10 days in the highest concentration of 2.5 mM. The median life span (LC 50 ) of the concentrations of 100 µM, 250 µM, 500 µM and 1 mM were 39 days, 38 days, 34 days and 30 days respectively for the early health phase fly ( Fig. 2A ) and 19 days, 18 days, 16 days and 15 days respectively for the transition phase fly ( Fig. 2B) . A comparison of survival curves of early health phase and transition phase showed a significant difference in response among all the tested concentrations (Log-rank [Mantel-Cox] Test, P < 0.0001). From this study, we carefully choose 100 µM, 250 µM, 500 µM and 1000 µM concentrations of CUR to understand the DA-ergic neuroprotective efficacy of CUR in the ROT-mediated life phase-specific fly PD model. All these concentrations of CUR did not affect the survival of the fly at the selected treatment windows. Care was also taken in such a way that selected CUR concentrations do not cause any mobility defects. Results of the negative geotaxis assay (NGA) illustrated that the selected CUR concentrations induced no mobility defects in the fly of the early health phase and transition phase ( Suppl. Fig. S1 ). 3.2 CUR rescues mobility defects mediated by ROT in both co- and pre-feeding regimens during the early health phase of Drosophila We assessed the negative geotaxis ability of the ROT-mediated adult early health phase fly PD model to decipher the mobility alterations under both the CUR pre- and co-feeding conditions. The distance that the fly climbed in 12 sec was assayed after 4 days and 5 days of exposure to 500 µM ROT or ROT along with CUR (100 µM, 250 µM, 500 µM and 1000 µM). After 4 days of feeding the fly with ROT, it exhibited slowness in movement as indicated by a significant decrease in the speed (bradykinesia), which is the characteristic clinical feature of PD in humans. The speed of the fly was significantly improved when ROT was fed along with CUR (co-feeding regime) as compared to the fly fed with ROT alone (Fig. 3 A). The mobility of ROT-fed fly was significantly reduced after 5 days whereas the CUR co-feeding rescues the phenotype as observed through the improved speed of the fly, suggesting the protective efficacy of curcumin (Fig. 3 B). Pre-feeding the fly with CUR for 2 days (Fig. 3 C & D ), 3 days (Fig. 3 E & F ) and 5 days (Fig. 3 G & H ) and exposed to ROT for 4 days and 5 days also showed similar rescue (significant protective effect at lower concentrations i.e. 100 and 250 µM indicates the resilience provided by CUR pre-feeding comparing to the co-feeding regimen), suggesting that observed protective efficacy of CUR is not through physical interaction and sequestration of the toxin. CUR per se has no adverse influence on the mobility performance of the flies. 3.3 CUR fails to rescue mobility defects mediated by ROT in both co- and pre-feeding regimens during the late health and the transition phases of Drosophila The adult life span of Drosophila is categorized into a health phase, transition phase and senescent phase [ 12 ]. It has been shown that there is a significant variation (about 23%) in genome-wide transcript profiles with age in Drosophila [ 11 ]. The efficacy of genotropic drugs depends on the availability of the target molecules at that stage of the life cycle. Hence, there is a possibility that targets of genotropic compounds such as CUR may not be present in all life stages [ 23 ]. To understand this paradigm, we tested CUR efficacy in the age groups of the late health phase (30 days old fly) and transition phase (50 days old fly). CUR could not improve the mobility defects induced by ROT in both the co- and pre-feeding regimens during the late health phase ( Suppl. Fig. S2 ) and transition phase (Fig. 4 ) of Drosophila as quantified with climbing ability. The distance that the fly climbed in 12 sec after one day and two days of exposure to 25 µM ROT or ROT along with CUR (100 µM, 250 µM, 500 µM and 1000 µM) was assayed. Feeding with ROT alone significantly impaired the mobility of the fly while feeding the fly with CUR (500 µM) per se did not show any effect on mobility. The co-feeding of CUR could not improve the mobility defects induced by ROT in both the late health phase ( Suppl. Fig. S2 ) and transition phase (Fig. 4 A & B). Pre-feeding the flies of the late health phase with CUR exposed to ROT for 1 day and 2 days also could not rescue the mobility defects during late health phase ( Suppl. Fig. S2) . Pre-feeding the flies with CUR for 2 days (Fig. 4 C & D ), 3 days (Fig. 4 E & F ) and 5 days (Fig. 4 G & H ) and exposed to ROT for 1 day and 2 days also showed no improvement in mobility during the transition phase of the fly. With far-reaching limitations of therapeutic efficacy in NDDs like PD, which shows an average onset at 50 years (according to http://www.ninds.nih.gov/disorders/parkinsons disease), CUR failed to rescue the mobility defects in both the pre- and co-feeding regimens during late health phase ( Suppl. Fig. S2 ) and transition phase (Fig. 4 ) as indicated by negative geotaxis assay. These results suggest the limitation of CUR as a therapeutic compound in late-onset NDDs such as PD. However, whether CUR can be a prophylactic agent is yet to be ascertained. 3.4 CUR rescues “neuronal dysfunction” during the early health phase but not during the transition phase In the Drosophila brain, the number of DAergic neurons (DNs) in different clusters viz. PAL, PPL1, PPL2, PPM1/2 and PPM3 are 5, 12, 6/7, 8/9 and 6 respectively ( Suppl. Fig. S3 ). The number of DNs was quantified by using fluorescently labelled secondary antibodies targeting against the primary antibody which is the rate-limiting enzyme in dopamine synthesis, Tyrosine hydroxylase (TH). The image of the Drosophila brain during the early health phase and transition phase captured through a fluorescence microscope is depicted in Fig. 5 A and Fig. 6 A respectively. Although there is a natural variation in the number of DNs of the same neuronal cluster, no significant difference is observed among the different treatment groups analyzed during the early health phase (Fig. 5 B) and transition phase (Fig. 6 B). The FI of the secondary antibodies targeting the primary antibody (anti- TH) is an indicator of the amount of TH synthesis. Hence, the diminished FI can be an indicator of the level of DA-ergic neurodegeneration. During the early health phase of the fly, the FI of the fly fed with 500 µM ROT for 5 days was significantly reduced by 42.36%, 42.55%, 42.31%, 45.11% and 44.89% in PAL, PPL1, PPL2, PPM1/2 and PPM3 respectively when compared to control as observed under the fluorescence microscope. Co-feeding the fly of the early health phase with CUR could significantly improve the FI (Fig. 5 C). In the case of transition phase fly, feeding with ROT alone led to significant reduction in the FI by 55.20%, 57.83%, 55.70%, 47.49%, and 56.74% in PAL, PPL1, PPL2, PPM1/2 and PPM3 respectively after 48 hr. Co-feeding the fly of the transition phase with CUR could not improve the reduction in FI (Fig. 6 C). The cluster-wise analysis of DA-ergic neuronal FI showed significant rescue in neuronal dysfunction in all the clusters during the early health phase but not during the transition phase of life stages of Drosophila . The total intensity sum of all the DNs in the fly brain was clubbed together for further analysis. Results reveal that ROT significantly downregulates the FI (~ 50%) (Suggesting diminished levels of TH protein synthesis) which could be significantly altered upon co-feeding with CUR during early health phase of the fly (Fig. 5 D). But the significantly reduced FI (~ 50%) of the DNs during the transition phase of the fly could not be improved upon co-feeding with CUR (Fig. 6 D). These results indicate that CUR can rescue the neurodegeneration induced by ROT only during the early health phase but not during the transition phase of the fly. The results from the present study support the findings of the negative geotaxis assay which indicated that CUR rescues mobility defects only during the early health phase but not during the transition phase. These results reveal that the synthesis of the TH for DA synthesis is downregulated as the FI of the neuron is directly proportional to the level of TH. Hence, the level of dopamine synthesis may be reduced in the brain. This is ascertained by quantifying the level of DA and its metabolites using HPLC-ECD. 3.5 Curcumin replenishes diminished DA and its metabolites (DOPAC and HVA) only during early health phase but not during transition phase and doesn’t influence DA turn over : Reduced DA-ergic neuronal FI is directly proportional to the diminished TH synthesis. Quantification of FI revealed that the intensity was significantly reduced under ROT-mediated conditions revealing the neurodegeneration which could be improved upon co-feeding with CUR during the early health phase but not during the transition phase of the fly. Also, CUR failed to improve the mobility defects induced by ROT in the transition phase fly (Fig. 4 ) . Hence, we asked whether the failure in improving the climbing ability during this phase is due to CUR’s failure in replenishing brain dopamine levels. To confirm this correlation, we estimated the levels of DA and its metabolites (DOPAC and HVA) in fly brain tissue extracts of different treatment groups under study using HPLC-ECD ( Suppl. Fig. S4 ). Feeding the fly with ROT alone led to a significant decrease in DA levels by 38% and 26% (Fig. 7 A), DOPAC levels by 38% and 13% (Fig. 7 B) and HVA levels by 29% and 21% (Fig. 7 C) during the early health phase and transition phase of the fly respectively. Co-feeding with 500 µM CUR and 1000 µM CUR could significantly increase the DA level by 17%, DOPAC levels by 21% and 18% and HVA levels by 12% and 14% respectively (Fig. 7 A,B,C) as compared to ROT-mediated group whereas lowest concentration of 250 µM CUR showed no significant difference in DA and HVA levels but improved the DOPAC level by 13% during the early health phase of the fly. Co-feeding the transition phase fly with CUR showed no improvement in the brain DA, DOPAC and HVA levels (Fig. 7 A,B,C) as compared to ROT-induced group suggesting that efficacy of CUR is life stage-specific in fly PD model. This result indicates that CUR can rescue DA, DOPAC and HVA levels thereby rescuing the neurodegeneration during the early health phase but fails during transition phase of the fly. The present study suggests that CUR-mediated modulation of perturbations in DA metabolism is restricted to the adult early health phase and not to the transition phase. This illustration suggests that the genetic targets and molecular networks of genotropic drug CUR-mediated correction process may not be active/expressive at optimum levels during later phases of the adult life. From this result, it can be hypothesized that CUR rescues the perturbations in brain DA metabolism in ALSS fashion. This critical observation underlines the limitations of CUR as a therapeutic agent in the late-onset NDDs such as PD. The quantification of DA and its metabolites during the early health phase of the fly upon treatment with 500 µM ROT leads to a significant decrease in brain-specific DA, DOPAC and HVA when compared with control. Since mitochondria are involved in several metabolic pathways, it is possible that the inhibition of mitochondrial complex I by ROT also affects the metabolism of DA and downstream catecholamines. In the case of the transition phase fly, the level of DA, DOPAC and HVA is significantly lowered under ROT-mediated conditions. Upon co-feeding with CUR, the level of DA, DOPAC and HVA could be significantly rescued during the early health phase but not during the transition phase of the fly. This could be due to the lack of targets of CUR during the transition phase of the fly. Upon comparing the rescue patterns with respect to control, results reveal that there is no significant difference in DA turn over in both the health and transition phase of the fly. However, trend shows a marginal slow-down in the catabolism of DOPAC to HVA during the transition phase of the fly (Fig. 8 ). 4. Discussion In most populations, 3–5% of PD is explained by genetic causes linked to known PD genes. However, the majority of patients (90–95%) have idiopathic or sporadic PD [ 26 ]. Further, epidemiological studies indicate a relationship between various environmental factors such as pesticide exposure and the development of sporadic PD in later stages of life [ 1 , 27 ]. Hence, we developed an adult life stage (health and transition phase) specific fly model of PD using mitochondrial complex I inhibitor neurotoxicant rotenone [ 5 ]. Adult life phase specific gene expression profile variation across model organisms suggest the relevance and importance of life phase-specific animal models for understanding the pathophysiology of late-onset NDDs such as PD and screening small molecules/nutraceuticals with potential neuroprotective efficacy and deciphering molecular basis of their biological activity. Studies on animal models have shown the neuroprotective efficacy of CUR [ 16 – 18 ]. Contradictory reports of concentration-dependent CUR toxicity have been reported in animal models [ 28 ] and in Drosophila model [ 23 ]. However, the toxicity of CUR in animal models is under-reported. Hence, we performed a comprehensive experiment with an array of CUR concentrations to identify the non-toxic concentrations of CUR in both the adult early health phase and transition phase of the Drosophila . Present experimental approach employing CUR was to expose the Drosophila to a range of concentrations to determine its detrimental effects and select a suitable range of non-toxic concentrations for further studies. We employed 8 concentrations ranging from 25 µM to 2.5 mM of CUR in both the adult early health phase and transition phase male Drosophila . It was found that CUR concentrations of 2.5 mM had a deleterious effect on the viability of the fly, whereas concentrations lower than 2.5 mM showed no observable toxicity in the early adult health phase fly up to 10 days ( Fig. 2 ). In the highest concentration of 2.5 mM, all the flies employed in the experiment were dead by the 30th day and 11th day of CUR treatment in the case of the early health and transition phases respectively. The concentration below 1 mM did not show any mortality up to 10 days of exposure to CUR, indicating that there was no toxicity in all the lower concentrations. CUR toxicity study in human also suggests that it primarily causes gastrointestinal disturbances, diarrhoea, as well as distension and gastroesophageal reflux disease [ 29 ]. Therefore, it is essential to make out the toxic concentrations of CUR before employing it as a neuroprotective agent in the fly model of PD. Hence, we picked up sub-lethal concentrations of CUR (100 µM, 250 µM, 500 µM and 1 mM) and the same concentrations were employed in life phase-specific ROT-mediated Drosophila model of PD under pre- and co-feeding regimen. To understand DA-ergic neurodegeneration and to decipher the effectiveness of therapeutic molecules, many laboratories either co-treat or pre-treat young PD fly models belonging to the health phase to screen small molecules/drugs/nutraceuticals and concluded about their DA-ergic neuroprotective efficiency [ 6 , 21 , 22 , 30 ]. They determined the neuroprotective efficacy of the molecules by assessing behavioral markers such as mobility defects, biochemical markers such as anti-oxidant enzyme levels and levels of brain DA and its metabolite levels, or cytological markers such as degeneration of DNs in the whole brain of young animals whereas late-onset NDDs such as PD onsets during the transition phase of adult life!! The present study indicates that feeding the fly of the adult early health phase with ROT alone adversely decreases mobility (~ 30%) which could be improved upon co-feeding with CUR. Feeding the fly with CUR per se did not show any mobility defects (Fig. 3 A & B ). Results indicate that in both the co-feeding (Fig. 3 A & B ) and pre-feeding regimens (Fig. 3 C-H), CUR could confer neuroprotection during the early health phase. It has been reported that exposure to ROT induces severe mobility defects in Drosophila and the same can be rescued upon co-feeding with CUR [ 30 ]. In the case of the late health phase fly, CUR fails to rescue the mobility defects in both the co- and pre-feeding regimens ( Suppl . Fig. S2 ). In the case of the transition phase fly, CUR fails to rescue ROT-mediated mobility defects in both the co- and pre-feeding regimens as determined through the negative geotaxis assay (Fig. 4 ). These results are in agreement with previous work from our lab, where CUR confers DA-ergic neuroprotection only during the early health phase but not during the transition stage [ 23 ]. However, in the present study, CUR fails even during the late health phase, which can be attributed to mitochondrial complex I inhibitor ROT specific neurotoxicity. Previous studies demonstrated the neuroprotective efficacy of CUR in ROT-mediated neurotoxicity in cell culture, young adult Drosophila and rat models and substantiated that CUR sequesters the intracellular and mitochondrial ROS levels and inhibits the caspase-3/caspase-9 activity [ 16 , 22 , 31 , 32 ]. These results suggest that CUR may be a potential therapeutic compound for PD intervention. However, all these studies used young animals belonging to the adult early health stage. The results from the present study suggest that CUR can rescue ROT-mediated mobility defects in ALSS fashion; meaning CUR confers neuroprotection only during the early health phase and not in the late health phase and transition phase of the adult life. The death of DNs is the characteristic pathological marker of PD. Hence, in order to decipher the extent of DA-ergic neurodegeneration under induced PD conditions and possible rescue by CUR, we quantified the DNs in the whole fly brain followed by characterization of DA “neuronal dysfunction” by quantifying the FI emanated from the fluorescently labelled secondary antibody targeted against the primary anti-TH antibody using fluorescence microscopy. The results illustrate that there is no variation in the number of DNs between the control and PD brains of Drosophila in both the health and the transition phases of adult life (Fig. 5 B & 6 B). This observation is consistent with the previous works from our lab [ 5 ] and other laboratories [ 25 , 33 , 34 ], which is logical in the light of “dying back” phenomenon. The issue of loss of DNs per se in fly brain has been thoroughly evaluated in multiple fly models of PD (both genetic and sporadic) and demonstrated that there is no structural loss of DA neurons, but only the synthesis of TH is diminished [ 25 ]. Hence, we went ahead with the quantification of the FI of TH-positive neurons. Results illustrate that the fly treated with ROT alone led to a significant decrease in FI to ~ 50% which could be significantly rescued by 25–30% upon co-feeding with CUR during the early health phase (Fig. 5 C) while the diminished FI could not be rescued by CUR during the transition phase (Fig. 6 C). We also analyzed the results by quantifying the FI of all the DNs present in the fly brain of different treatment groups (Fig. 5 D & 6 D). Similar results were obtained when analyzed in treatment group-wise. As the FI of the secondary antibodies is directly proportional to the level of synthesis of the TH, diminished levels of FI suggest the decreased levels of TH. Hence, it is possible that the level of DA synthesis might be reduced in the fly brain. This approach allowed us to accurately assess the extent of neurodegeneration of DNs in the fly PD models. This result is further substantiated by quantifying the levels of the brain-specific neurotransmitter DA and its metabolites (DOPAC and HVA) using HPLC-ECD. The present study shows that feeding the fly with ROT alone led to a significant reduction in the levels of DA, DOPAC and HVA during the early health and the transition phases of Drosophila (Fig. 7 ). Similar results showing significant reduction in DA, DOPAC and HVA levels are indicated in 10 days old young fly [ 22 ], young mice [ 35 ], young wistar rat [ 36 ]. All these results indicate that ROT significantly downregulates the DA level under induced PD conditions. The present study demonstrates that diminished levels of DA, DOPAC and HVA upon ROT treatment could be significantly rescued through co-feeding with CUR during the early health phase but not during the transition phase of the adult fly (Fig. 7 ). Study on young 12 weeks old albino wistar rat showed a significant reduction in DA and DOPAC levels upon ROT-mediated conditions which could be significantly improved upon co-feeding with CUR [ 36 ]. Another study indicated that feeding Danshensu (a plant extract; 60mk/kg for 28 days) could significantly improve the levels of DA, DOPAC and HVA in ROT-induced young 12 weeks old C57BL/6 mice indicating that the levels of DOPAC and HVA are proportional to DA content [ 35 ]. CUR is shown to restore the levels of DA, DOPAC and HVA in neurodegeneration mediated by lipopolysaccharide in young Sprague-Dawley rats [ 37 ]. All these results support the present study and indicate that CUR restores the levels of DA, DOPAC and HVA in ROT-mediated PD models. However, these studies are restricted to 5–20 days young fly [ 6 , 22 ] and 10–12 weeks young mice and rat models [ 35 , 36 ] whose age is comparable with the early health phase of adult life. There is no data available relating to the levels of DA and metabolites in PD models of the transition phase of adult life, during which PD sets in. This is the first detailed study to have shown the levels of DA and its metabolites in the transition phase of Drosophila model of ROT-mediated PD. Present study reveals that CUR does not influence the DA turn over during both the health and the transition phases of adult life. Research on DA metabolism gives an insight into the biochemical changes in DA-ergic system associated with PD. However, the biological significance of “turnover ratio” which denotes the level of metabolites (DOPAC and HVA) with respect to DA is not discussed in many studies quantifying DA and metabolites. Pagan et al. [ 38 ] reported DA metabolites as “exploratory biomarkers” with reference to CSF levels of DOPAC and HVA. Although, compiling the CSF levels of HVA with xanthine improves its use as a biomarker; CSF levels of HVA poorly correlate with PD severity or progression [ 39 ]. Though DOPAC is the primary metabolite of DA [ 40 ], LeWitt [ 41 ] commented that representing the CSF level of DOPAC as a marker for the progression of PD is not valid though it shows severity. A significant amount of DOPAC and HVA, albeit lower DA levels, is seen in the human brain wherein DA-ergic innervation has not been implicated [ 42 ]. Furthermore, the levels of neurotransmitters in CSF partially implicate the metabolism indicating limited relevance to neurotransmission in the brain. Hence, quantitative analysis of brain-specific DA and its metabolites concentrations are unrelated to DA-ergic neurotransmission which could be due to the possible role of capillary walls and glial cells in the catabolism of DA that needs to be further investigated [ 42 ]. Hence, the notion “turnover” is not applicable with respect to the activity of DA-ergic system [ 42 ]. These findings suggest that CUR-mediated nourishment of DA metabolism is restricted to the adult health phase and not to the transition phase. This illustration indicates that genetic targets and molecular networks of genotropic drug CUR-mediated correction process may not be active/expressive at optimum levels during later phases of the adult life. However, there is no significant difference in the DA turn over pattern in both the health and transition phase of the fly but trend shows a marginal slow-down in the catabolism of DOPAC to HVA during the transition phase of the fly (Fig. 8 ). Thus the study underlines the limitation of CUR as a therapeutic agent in late-onset NDDs such as PD. Figure 9 illustrates a detailed schematic presentation of our proposed study model. The present study provides insights into the underlying reasons for neuroprotective efficacy of CUR during the adult health phase of Drosophila . Further, it also provides insights into the probable cause for the inefficacy of CUR during the adult transition phase of Drosophila . It is important to figure out why CUR is failing to sustain the activity of the TH during the transition phase, understanding of which will have critical implications in developing therapeutic strategies for late-onset neurodegenerative diseases such as PD. Figuring out the underlying molecular networks in an ALSS fashion will be a path-breaking contribution in addressing the pathophysiology of PD. This is the first report to decipher the fact that CUR has limitations as a therapeutic molecule in the ROT-mediated idiopathic Drosophila model of PD, and its efficacy is restricted only to the early health phase of adult life. 5 Conclusion The present study reveals that CUR mitigates the mobility defects induced by ROT during the early health phase but fails during the late health and transition phase in both the co- and pre-feeding regimens in the adult Drosophila model of sporadic PD. Analysis of DNs reveals that their number remains unchanged under ROT-induced PD conditions but the FI of the secondary antibody targeted against the primary anti-TH antibody is significantly reduced suggesting the diminished levels of TH synthesis. The reduction in the FI could be improved upon co-feeding with CUR only during adult early health phase but not during the transition phase of the fly. Further analysis of the brain-specific DA and its metabolites reveals that CUR rescues DA and its metabolites during the early health phase only. The present study explains that curcumin’s ability to modulate perturbed DA metabolism is constrained to the adult early health phase. Observed contradictions can be attributed to the availability of genetic targets of genotropic nutraceutical curcumin in an adult life phase-specific fashion. Hence, it confers DA-ergic neuroprotection in adult early health phase but not in transition phase. Declarations Declaration of competing interest The authors declare that there is no conflict of interest. Author Contribution M. Ayajuddin: Data curation; Formal analysis; Investigation; Methodology; Software; Visualization; Roles/Writing - original draft; and Writing - review & editing. A. Das: Formal analysis; Methodology; Software; Visualization; Data curation; Writing - review & editing. S.C. Yenisetti: Conceptualization; Methodology; Funding acquisition; Project administration; Resources; Supervision; Validation; Visualization; Writing - review & editing; and Data curation. Acknowledgement This research is supported by the Department of Biotechnology (DBT), India (R&D grant no. BT/405/NE/U-Excel/2013) awarded to SCY. Part of the work was presented at the International Conference on Recent Advances in Biotechnology and Environmental Science, organised by Vellore Institute of Technology (VIT), Vellore, India, 16-18 December, 2022; 4th International Conference on Neurology and Brain Disorders (INBC-2021), 9-10 September, 2021, Italy, Rome by MA (for which MA was awarded with international travel fellowship by IBRO (International Brain Research Organization, France); and at International Conference on Neuroscience and Neurological Disorders and 37th Annual Meeting of Society for Neurochemistry (SNCI), India, organised by Department of Biomedical Engineering, North Eastern Hill University, Shillong, Meghalaya, India. 14-16 September, 2023 by SY (plenary talk). MA received DBT (Department of Biotechnology, India)-JRF (junior research fellowship) and SRF (Senior research fellowship) from ICMR (Indian Council of Medical Research), India. AD received JRF and SRF from DBT. Data Availability The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. References Saleh, M.A., Amer-Sarsour, F., Berant, A., Pasmanik-Chor, M., Kobo, H., Sharabi, Y., et al. Chronic and acute exposure to rotenone reveals distinct Parkinson's disease-related phenotypes in human iPSC-derived peripheral neurons. Free Radic Biol Med. 213, 164–173 (2024). Pouchieu, C., Piel, C., Carles, C., Gruber, A., Helmer, C., Tual, S., et al. Pesticide use in agriculture and Parkinson's disease in the AGRICAN cohort study. Int. J. Epidemiol. 47 (1), 299–310 (2018). Furlong, M., Tanner, C.M., Goldman, S.M., Bhudhikanok, G.S., Blair, A., Chade, A., et al. Protective glove use and hygiene habits modify the associations of specific pesticides with Parkinson's disease. Environ. 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J Neurochem. 48(6), 1725–1729 (1987). Additional Declarations No competing interests reported. Supplementary Files SupplmaterialSciRep..Revised.docx floatimage9.jpeg 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-4645640","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":329605906,"identity":"d6fac436-d5e2-440f-a505-f31b1b3cf2d3","order_by":0,"name":"Mohamad Ayajuddin","email":"","orcid":"","institution":"Nagaland University","correspondingAuthor":false,"prefix":"","firstName":"Mohamad","middleName":"","lastName":"Ayajuddin","suffix":""},{"id":329605907,"identity":"50c3fad9-8a7e-4370-9e5d-4afa1c7034a8","order_by":1,"name":"Abhik Das","email":"","orcid":"","institution":"Nagaland University","correspondingAuthor":false,"prefix":"","firstName":"Abhik","middleName":"","lastName":"Das","suffix":""},{"id":329605910,"identity":"c41491d5-5e3d-4dda-abcb-701cbc767665","order_by":2,"name":"Sarat Yenisetti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIiWNgGAWjYHACNiBmBjEYHwAZPOzNBxgYEgqI08JsANLCcywBqMWAOC1sEiAGWAsDHi0G5w8/e/Bzh3U+/4z0Z9UVFdYyPGzciR8eGDDI84sdwK7lRpq5Ye+ZdMsZN3LMbp45k87Dw8a7WQLoMMOZsxNwaOFhk+BtO2zAcCOH7WZj22Eee/neDSAtCQa3cWg5f4ZN8i9Qi/yN9GeFjf8Og235gVfLgRw2aZAtBjcSzBgbG8BatuG1RfJGmpm0bFu6geGZN8aSDcfAftlmkWAggdMvfMAQk3zbZm0gdzz94ceGGmt7kMNu/qiwkeeXxq5F4QCMJYCqQAKrchCQb4Cx+A/gVDQKRsEoGAUjHAAA+A9b/gdHxXwAAAAASUVORK5CYII=","orcid":"","institution":"Nagaland University","correspondingAuthor":true,"prefix":"","firstName":"Sarat","middleName":"","lastName":"Yenisetti","suffix":""}],"badges":[],"createdAt":"2024-06-27 03:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4645640/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4645640/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61233781,"identity":"831ead47-a15f-4847-84bf-0ec12380f7e6","added_by":"auto","created_at":"2024-07-27 18:13:30","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":235656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-feeding and pre-feeding regimen using \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e: (A) \u003cstrong\u003eCo-feeding\u003c/strong\u003e: Flies belonging to different life phases (5 days: Early health span; 30 days: Late health span; and 50 days: transition phase) were fed with ROT alone or ROT and CUR for 5 days in case of early health phase and 2 days in case of late health phase and transition phase flies. Control fly remained in 5% sucrose only while the CUR (500 µM) \u003cem\u003eper se\u003c/em\u003e group was fed with CUR only. The negative geotaxis assay (NGA) was performed on the 4\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e day of exposure to ROT in the case of early health phase fly and on 1\u003csup\u003est\u003c/sup\u003e and 2\u003csup\u003end\u003c/sup\u003e day of exposure to ROT in case of the late health phase and transition phase flies respectively. (B) \u003cstrong\u003ePre-feeding regimen\u003c/strong\u003e: Flies of different life stages (Early health phase; late health phase; and transition phase) were pre-fed with CUR for 3 days. Control and the group to be treated with ROT remain in 5% sucrose during this period. The fly was then exposed to ROT for 5 days in case of the early health phase and 2 days in case of the late health phase and transition phase flies. Control and CUR (500 µM) per se group remain in 5% sucrose. NGA was performed on 4\u003csup\u003eth\u003c/sup\u003e and 5\u003csup\u003eth\u003c/sup\u003e day after exposure to ROT in case of the early health phase fly and on 1\u003csup\u003est\u003c/sup\u003e and 2\u003csup\u003end\u003c/sup\u003e day of exposure to ROT in case of the late health phase and transition phase flies.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/ff9b7f56afc8acc7e54f48ee.jpeg"},{"id":61233600,"identity":"e4fa40f0-90b7-45d4-be47-cac3fd268b3c","added_by":"auto","created_at":"2024-07-27 18:05:30","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":58166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDose- and time-dependent mortality of OK male fly exposed to CUR during different phases of adult life:\u003c/strong\u003eMortality pattern among adult male flies of early health phase (\u003cstrong\u003eA\u003c/strong\u003e) and transition phase (\u003cstrong\u003eB\u003c/strong\u003e), exposed to eight different concentrations of CUR (25 µM, 50 µM, 100 µM, 250 µM, 500 µM, 1 mM, 1.5 mM and 2.5 mM) showed concentration-dependent lethality. Mortality data were collected every 24 Hr for each group till all the flies were dead. A comparison of survival curves showed a significant difference in response among different tested concentrations (log-rank [Mantel–Cox] test, p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/02c58173189ebc8fdd9cdd87.jpeg"},{"id":61233599,"identity":"1804c640-fe9d-4161-b1c0-5dd414b6e3ee","added_by":"auto","created_at":"2024-07-27 18:05:30","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":214531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCurcumin rescues rotenone induced mobility defects during the early health phase of adult life in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e model of PD\u003c/strong\u003e.\u0026nbsp; Negative geo-taxis assay of early health phase fly in co-feeding regime (A \u0026amp; B) and pre-feeding\u0026nbsp; (C – H) regimens: In the co-feeding regimen, fly was fed with ROT alone or ROT along with CUR for 5 days the mobility defect was assessed on 4\u003csup\u003eth\u003c/sup\u003e day (A) and 5\u003csup\u003eth\u003c/sup\u003e day (B). In the pre-feeding regimen, fly was pre-fed with CUR alone for 2 days (C \u0026amp; D), 3 days (E \u0026amp; F) and 5 days (G \u0026amp; H) and exposed to ROT for 5 days and the mobility defects were assessed on the 4\u003csup\u003eth\u003c/sup\u003e day (C, E \u0026amp; G) and 5\u003csup\u003eth\u003c/sup\u003e day (D, F and H). CUR rescues mobility defects during the adult early health phase in both the co-feeding and pre-feeding regimens in the fly model of ROT-mediated sporadic PD. (One way ANOVA followed by Newman-Keuls Multiple Comparison Test showed significant difference in mobility. * P\u0026lt;0.05; ** P\u0026lt;0.001; *** P\u0026lt;0.0001 compared with ROT-treated group)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/7959175ca940999d0901a2f4.jpeg"},{"id":61233783,"identity":"16b5a3d2-4245-4b6f-a715-14b80c02aacf","added_by":"auto","created_at":"2024-07-27 18:13:30","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":888046,"visible":true,"origin":"","legend":"\u003cp\u003ev\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/324e3398d9144c1817ef400f.jpeg"},{"id":61233851,"identity":"8f202b11-ee86-4463-8578-46f41f9cc93d","added_by":"auto","created_at":"2024-07-27 18:21:30","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":296123,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization and quantification of DA-ergic neurodegeneration in the whole fly brain of the early health phase through anti-TH antibody immunostaining\u003c/strong\u003e: Characterization of dopaminergic neurodegeneration in the fly brain of the early health phase \u003cstrong\u003e(A). \u003c/strong\u003eThe scale bar of all the images in the panel is 20 µm. (CTR- Control; ROT- Treated with ROT only; R500- ROT along with CUR 500 µM concentration; R1000- ROT along with CUR 1000 µM concentration; Represented images are “merged” Z-stacking images; however, the quantification of DA-ergic neuronal number and FI is performed in 3D Z-stack images).\u003cstrong\u003e \u003c/strong\u003eQuantification of DNs reveals that there is no loss in the number of DNs upon treatment with ROT alone or ROT along with multiple concentrations of CUR \u003cstrong\u003e(B)\u003c/strong\u003e. However, ROT leads to “neuronal dysfunction” as characterized by quantification of DA-ergic neuronal FI that is proportional to the amount of TH protein which could be rescued upon therapeutic intervention of CUR \u003cstrong\u003e(C)\u003c/strong\u003e. Further, a similar trend is observed upon pooling the FI sum of all the clusters in the fly brain \u003cstrong\u003e(D)\u003c/strong\u003e suggesting that CUR intervention can rescue the depleted levels of TH during the adult early health phase of fly. (One way ANOVA followed by Newman-Keuls Multiple Comparison Test. *p\u0026lt;0.05; **p\u0026lt;0.001; ***p\u0026lt;0.0001; NS- Not significant).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/16e0dd2e6eda032660e7326e.jpeg"},{"id":61233784,"identity":"b8c9b18b-1a91-4496-a4bb-a588e746a8ab","added_by":"auto","created_at":"2024-07-27 18:13:30","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":662225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization and quantification of DA-ergic neurodegeneration in the whole fly brain of the transition phase through anti-TH antibody immunostaining\u003c/strong\u003e: Characterization of dopaminergic neurodegeneration in the fly brain of the transition phase \u003cstrong\u003e(A). \u003c/strong\u003eThe scale bar of all the images in the panel is 20 µm. (CTR- Control; ROT- Treated with ROT only; R500- ROT along with CUR 500 µM concentration; R1000- ROT along with CUR 1000 µM concentration; Represented images are “merged” Z-stacking images; however, the quantification of DA-ergic neuronal number and FI is performed in 3D Z-stack images).\u003cstrong\u003e \u003c/strong\u003eQuantification of DNs reveals that there is no loss in the number of DNs upon treatment with ROT alone or ROT along with multiple concentrations of CUR \u003cstrong\u003e(B)\u003c/strong\u003e. However, ROT leads to “neuronal dysfunction” as characterized by quantification of DA-ergic neuronal FI that is proportional to the amount of TH protein which could not be rescued upon therapeutic intervention of CUR \u003cstrong\u003e(C)\u003c/strong\u003e. Further, a similar trend is observed upon pooling the FI sum of all the clusters in the fly brain \u003cstrong\u003e(D)\u003c/strong\u003e suggesting that CUR intervention cannot rescue the depleted levels of TH during the transition phase of fly suggesting its limitation as a therapeutic agent in late-onset NDDs such as PD. (One way ANOVA followed by Newman-Keuls Multiple Comparison Test. NS- Not significant).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/6a9e1bfc0db674683a7542a6.jpeg"},{"id":61233605,"identity":"72248d58-73b7-44a6-95b6-8426deefe038","added_by":"auto","created_at":"2024-07-27 18:05:30","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":303244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCurcumin replenishes diminished levels of brain specific DA and its metabolites only during the adult early health phase\u003c/strong\u003e: Feeding the fly with ROT alone led to a significant reduction in brain DA (A), DOPAC (B) and HVA (C) levels during the early health phase and transition phase but co-feeding with CUR could significantly alter the diminished DA, DOPAC and HVA levels only during the early health phase of the fly. However CUR does not influence the DA turn over in both the adult life phase of the fly (D). (Two-way ANOVA followed by Bonferroni post-tests; *p\u0026lt;0.05; **p\u0026lt;0.01; NS- Not significant; compared with ROT treated group).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/168da0dd979ea667955a061a.jpeg"},{"id":61233606,"identity":"60027e59-2b68-40ab-8f28-1f7d44c958fe","added_by":"auto","created_at":"2024-07-27 18:05:30","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":351377,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of alterations in levels of players involved in brain DA metabolism under nutraceutical curcumin mediated intervention strategy in ROT-mediated \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDrosophila\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e model of PD\u003c/strong\u003e: DA and its metabolites- DOPAC and HVA are significantly reduced upon ROT-induced PD condition which could be altered upon co-feeding with CUR during the early health phase but not during the transition phase (CTR: Control; TD: Treated with ROT alone; RES: CUR mediated rescue, if any).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/20deb45ad9369c48ab3c3888.jpeg"},{"id":62141176,"identity":"a336965b-383c-4ea5-ade2-17fd22348f6d","added_by":"auto","created_at":"2024-08-09 17:18:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4032548,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/ce12cd32-d815-4ab7-ab99-a673b75db495.pdf"},{"id":61233597,"identity":"96d2f93c-9fae-48cf-b447-fa3e7b66de4f","added_by":"auto","created_at":"2024-07-27 18:05:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":859040,"visible":true,"origin":"","legend":"","description":"","filename":"SupplmaterialSciRep..Revised.docx","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/99360ac44437a3d789f3c420.docx"},{"id":61233852,"identity":"b63ef48e-2214-4aae-b297-6e0260f347d5","added_by":"auto","created_at":"2024-07-27 18:21:30","extension":"jpeg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":114560,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4645640/v1/7e06e7a68a3cd9fb5154e1e8.jpeg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Age specific neuroprotection of curcumin is through differential modulation of brain dopamine metabolism: Insights from Drosophila model of Parkinson’s disease ","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a movement disorder suffering among 1% of the population worldwide. Epidemiological and animal model studies suggest a strong correlation between the onset of PD and exposure to pesticides and other environmental toxins [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Several animal models of PD have been developed using the herbicide rotenone as a neurotoxicant [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Rotenone can cross the blood brain barrier, impair the oxidative phosphorylation of the electron transport system (ETC) of mitochondria generating reactive oxygen species (ROS) and further inducing apoptosis and cytotoxicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] leading to the pathological characteristics of PD including dopaminergic (DAergic) neurodegeneration, depletion of dopamine levels in the brain, and mitochondrial dysfunction [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\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=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The adult life stages of \u003cem\u003eDrosophila\u003c/em\u003e is 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=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These life stages of model organisms such as mice and fly, are characterized by different patterns of gene expression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], parallel with the equivalent life stages of humans. For instance, 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=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A large number of stage-associated pathways independently influence a common and unique complex process of the life stages of \u003cem\u003eDrosophila\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. All these studies emphasize the importance and necessity of developing life stage-specific animal models for late-onset NDDs such as PD. Hence, our laboratory made a comprehensive effort and recently developed a neurotoxin rotenone (which inhibits mitochondrial complex I of ETC) induced adult life stage-specific (ALSS) \u003cem\u003eDrosophila\u003c/em\u003e model of sporadic PD [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This novel model is a good tool to understand the progression of pathophysiology and to screen potential therapeutic compounds/nutraceuticals and identify their molecular targets of activity, knowledge of which will assist in developing novel therapeutic strategies for 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=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Researchers have suggested the efficacy of natural products by demonstrating their efficacy to modulate biochemical markers, anti-oxidant enzymes and phenotypes associated with the disease in different animal models. Their varied natural role in organisms overlay 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 (CUR) is an extensively investigated phytochemical with 23,314 PUBMED articles, among which 316 articles are on PD (as on 15/04/2024). It possess powerful anti-inflammatory and anti-oxidant properties thereby exhibits significant neuroprotective properties by modulating neuro-inflammatory pathways, scavenging reactive oxygen species, and inhibiting the production of pro-inflammatory cytokines [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The therapeutic efficacy of CUR has been demonstrated in various diseases including PD [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Randomized clinical trials comprising 631 patients with various diseases have shown the beneficial role of CUR/turmeric [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Thus, CUR is one such potential candidate that can be explored for a therapeutic approach to several human diseases including NDDs like PD. However, 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=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This pattern is comparable to the corresponding life phase in mice, fly and humans. Nevertheless, several studies have shown the neuroprotective effects of CUR by employing young animal models of the adult health phase [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, while screening nutraceuticals for their DA-ergic 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=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur laboratory has previously demonstrated the ALSS neuroprotective efficacy of CUR in paraquat-mediated \u003cem\u003eDrosophila\u003c/em\u003e model of PD and also dose-dependent lethality of CUR \u003cem\u003eper se\u003c/em\u003e in \u003cem\u003eDrosophila\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Further, our laboratory recently developed adult health and transition stage specific PD model by inhibiting brain mitochondrial complex I using environmental neurotoxicant rotenone [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This model is closer to human PD condition and in order to validate the model we employed curcumin and demonstrated that its efficacy is ALSS. Here, we made an effort to understand the neuroprotective efficacy of CUR in ROT-mediated health and transition phase-specific \u003cem\u003eDrosophila\u003c/em\u003e model of PD by employing four different concentrations of CUR (100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1000 \u0026micro;M) in both the pre- and co-feeding regimen through assessing mobility phenotypes and further by characterizing the DA-ergic neurodegeneration/protection and modulation of DA metabolism and possible rescue/no rescue under induced PD conditions. For first time, we have demonstrated that time tested nutraceutical CUR has limitations to its therapeutic efficacy for late-onset NDDs such as PD and ALSS dopaminergic neuroprotective efficacy of CUR is mediated through differential regulation of brain dopamine metabolism and 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":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Fly husbandry\u003c/h2\u003e \u003cp\u003eFly was cultured according to the protocol described in Ayajuddin et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\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 were used in the present study. The flies were raised at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u003csup\u003e0\u003c/sup\u003eC with 12 Hour (Hr) 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 (Phom et al., 2014). For collecting the flies, they were mildly anaesthetized 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. Four-five days old flies were used for further experiment while late health span and transition phase flies were kept transferring routinely 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\u003e2.2 Chemicals\u003c/h2\u003e \u003cp\u003eThe required chemicals viz., Rotenone (Sigma, Cat: R8875), Curcumin (Sigma, Cat: 1386) and DMSO (Sigma, Cat: D8418) were used for feeding procedures. Standard dopamine (DA; Sigma-Aldrich, Cat: H8502) and its metabolites- 3,4-Dihydroxyphenylacetic acid (DOPAC; Sigma-Aldrich, Cat: 11569) and Homovanilic acid (HVA; Sigma-Aldrich, Cat: 69673) were used for quantifying DA and metabolites. Paraformaldehyde (Sigma, Cat: I58127), Triton X-100 (Sigma, Cat: T8787), Normal Goat Serum (NGS; Vector Lab, Cat: S1000), VECTASHIELD mounting medium (Vector Labs, CA, USA, Cat: H1000), Rabbit anti-Tyrosine hydroxylase (anti-TH) polyclonal primary antibody (Millipore, MA, USA, Cat: Ab152) and Goat anti-rabbit IgG H\u0026amp;L (TRITC labelled) polyclonal secondary antibody (Abcam, MA, USA, Cat: Ab6718) were used for immunostaining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Negative geotaxis assay\u003c/h2\u003e \u003cp\u003eThe upward mobility of the flies, also called negative geotaxis assay, was assessed as described in Ayajuddin et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In short, a single fly aspirated out of the vial was released into a plastic tube and acclimatized for 2 minutes. After gently tapping the fly to the bottom of the tube, the distance it could climb in 12 seconds was recorded. The same fly was given three chances and a minimum of 12 flies were noted for each concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Curcumin toxicity assay\u003c/h2\u003e \u003cp\u003eFour to five days and 50 days old \u003cem\u003eDrosophila\u003c/em\u003e males were fed on eight different concentrations of curcumin (25 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M, 1 mM, 1.5mM and 2.5 mM). Mortality was recorded every 24 Hr for 10 days. The concentrations at which no mortality was observed during this period were selected for further study to understand the protective efficacy of this molecule. Control flies were fed on 5% sucrose.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Curcumin pre- and co-feeding regimens\u003c/h2\u003e \u003cp\u003e For understanding the efficacy of CUR in the \u003cem\u003eDrosophila\u003c/em\u003e model of PD, two treatment regimens were employed i.e., Co-feeding regime and Pre-feeding regime. The concentrations of ROT used for the experiments were 500 \u0026micro;M, 25 \u0026micro;M and 10 \u0026micro;M for early health phase, late health phase and transition phase flies respectively. All these concentrations were selected as described in Ayajuddin et al. (2022). CUR concentrations were selected as described in the above section.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1\u003c/b\u003e: \u003cb\u003eCo-feeding and pre-feeding regimen using\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e: (A) \u003cb\u003eCo-feeding\u003c/b\u003e: Flies belonging to different life phases (5 days: Early health span; 30 days: Late health span; and 50 days: transition phase) were fed with ROT alone or ROT and CUR for 5 days in case of early health phase and 2 days in case of late health phase and transition phase flies. Control fly remained in 5% sucrose only while the CUR (500 \u0026micro;M) \u003cem\u003eper se\u003c/em\u003e group was fed with CUR only. The negative geotaxis assay (NGA) was performed on the 4th and 5th day of exposure to ROT in the case of early health phase fly and on 1st and 2nd day of exposure to ROT in case of the late health phase and transition phase flies respectively. (B) \u003cb\u003ePre-feeding regimen\u003c/b\u003e: Flies of different life stages (Early health phase; late health phase; and transition phase) were pre-fed with CUR for 3 days. Control and the group to be treated with ROT remain in 5% sucrose during this period. The fly was then exposed to ROT for 5 days in case of the early health phase and 2 days in case of the late health phase and transition phase flies. Control and CUR (500 \u0026micro;M) per se group remain in 5% sucrose. NGA was performed on 4th and 5th day after exposure to ROT in case of the early health phase fly and on 1st and 2nd day of exposure to ROT in case of the late health phase and transition phase flies.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(A) Co-feeding regime\u003c/b\u003e: In the co-feeding regimen, the fly was fed with ROT alone for ROT treatment group and a combination of ROT along with CUR (100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1000 \u0026micro;M) for the co-feeding group while the control fly remain in 5% sucrose only.\u003c/p\u003e \u003cp\u003e \u003cb\u003e(B) Pre-feeding regime\u003c/b\u003e: In the pre-feeding regimen, fly was fed with CUR (100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1000 \u0026micro;M) for 2, 3 and 5 days and then switched to ROT. For the ROT treatment group, fly was fed with 5% sucrose for 2, 3, 4 and 5 days and then transferred to ROT while the control fly was fed with 5% sucrose only. Climbing ability was assessed on the 4th and 5th day of feeding with ROT for the early health phase; while it was on 1st and 2nd day for the late health span and transition phase flies (\u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e).\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\u003e2.6 Quantification of dopaminergic neurodegeneration and tyrosine hydroxylase synthesis in whole fly brain using fluorescence microscopy:\u003c/h2\u003e \u003cp\u003eQuantification of dopaminergic neurons and fluorescence intensity (FI) was done according to Chaurasia et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Briefly, Briefly, the male OK flies were fixed in 4% paraformaldehyde (PFA) containing 0.5% Triton (TX)-100, at room temperature for 2 Hr, and then washed 5 times after every 15 minutes in phosphate-buffered saline with 0.1% TX-100 (PBST), at room temperature (RT). Blocking was done using 0.5% TX-100 and 5% NGS for 120 minutes at RT. Then, the brains were incubated in primary antibody (anti-TH) diluted in 1:250 ratio for 72 Hr at 4\u0026deg;C. The excess primary antibody was removed by washing the brains for 5X15 minutes in PBST. Brains were then incubated with a 1:250 dilution of secondary antibody (TRITC labelled) for 24 Hr at room temperature under dark conditions. After thorough washing for 5X15 minutes in PBST, brains were mounted in VECTASHIELD mounting medium and image acquisition was done on the same day.\u003c/p\u003e \u003cp\u003eThe idea to quantify the FI of fluorescently labelled secondary antibodies (using Carl Zeiss, ZEN 2012 SP2 software) was adapted from the protocol of Navarro et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], where they quantified the FI of GFP reporter. In brief, prepared/stained brains were viewed under a fluorescence microscope. Rhodamine filter was used for scanning the image. For image acquisition at 40x, red dot test for visibility of neurons was done for all the brains. Z-stack programming with constant intervals was performed. For image processing, on method column, image subset and maximum intensity projection (MIP) with X-Y Plane were created. From 3D 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 and a line was drawn around the neuron using a draw spline contour from graphics tools and the intensity sum was created in .xml format. The procedure was repeated for all the neurons in different clusters. Care was taken to select the fly brains with the same orientation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Quantification of brain dopamine and its metabolites using HPLC-ECD\u003c/h2\u003e \u003cp\u003eBrain-specific dopamine and its metabolites were quantified using High-Performance Liquid Chromatography (HPLC-Thermo Scientific, Dionex Ultimate 3000) equiped with an electrochemical detector (ECD) as described in Ayajuddin et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Briefly, the brains were homogenized in ice-chilled PBS. The supernatant collected after centrifugation was mixed with 5% TCA in a ratio of 1:1. 50 \u0026micro;L of the supernatant was kept aside for protein quantification before mixing with TCA. 20 \u0026micro;L of standard DA, HVA, DOPAC and 50 \u0026micro;L of the sample were loaded onto HPLC for quantification. MD-TM from Thermo Scientific (Cat: 701332) was used as the mobile phase and MCM 15 cm X 4.6 mm, 5 \u0026micro; C-18 packed column (Thermo Scientific, Cat: 70\u0026ndash;0340) was used as a stationary phase for elution of the monoamines. Inside the primary ECD containing two cells, detection of the monoamines was done at a range of -175 mV to +\u0026thinsp;225 mV as reduction and oxidation potential respectively. The third cell which is part of the Omnicell (secondary ECD module) was set at +\u0026thinsp;500 mV to reduce background noise. The data collection rate was set at 5 Hz. Chromatograms were analyzed using Chromaleon\u0026reg; 7, Thermo Scientific, United States\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Data analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed 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, one-way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test was performed. P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 CUR toxicity studies: \u003cem\u003eDrosophila\u003c/em\u003e is susceptible to CUR in a time-dose-dependent manner\u003c/h2\u003e \u003cp\u003e In order to understand the susceptibility of \u003cem\u003eDrosophila\u003c/em\u003e to CUR, male OK flies of early health and transition phase were fed with different concentrations of CUR (25 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M, 1 mM, 1.5 mM and 2.5 mM) while the control flies remain on 5% sucrose only. The survivorship of flies was observed and recorded every 24 Hr till all the flies were dead. A total of 100 flies were used for each concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2\u003c/b\u003e: \u003cb\u003eDose- and time-dependent mortality of OK male fly exposed to CUR during different phases of adult life\u003c/b\u003e: Mortality pattern among adult male flies of early health phase (\u003cb\u003eA\u003c/b\u003e) and transition phase (\u003cb\u003eB\u003c/b\u003e), exposed to eight different concentrations of CUR (25 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M, 1 mM, 1.5 mM and 2.5 mM) showed concentration-dependent lethality. Mortality data were collected every 24 Hr for each group till all the flies were dead. A comparison of survival curves showed a significant difference in response among different tested concentrations (log-rank [Mantel\u0026ndash;Cox] test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eCUR exerts concentration-dependent lethality on both the life stages of the fly. The survivability of the flies in the concentrations of 25 \u0026micro;M, 50 \u0026micro;M, 100 \u0026micro;M and 250 \u0026micro;M were very close to the control group. The concentrations of CUR ranging from 25 \u0026micro;M to 1 mM showed no observable toxicity while concentrations of 1.5 mM and 2.5mM affect the viability of the transition phase fly showing that higher concentrations of CUR could be harmful to the fly. The fly in the lowest concentration of 25 \u0026micro;M could survive for 55 days (maximum life span) while it could survive for 30 days in the highest concentration of 2.5 mM in the case of the early health phase fly. In the case of the transition phase fly, it could survive for only 30 days in the lowest concentration of 25 \u0026micro;M and 10 days in the highest concentration of 2.5 mM. The median life span (LC\u003csub\u003e50\u003c/sub\u003e) of the concentrations of 100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1 mM were 39 days, 38 days, 34 days and 30 days respectively for the early health phase fly (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e) and 19 days, 18 days, 16 days and 15 days respectively for the transition phase fly (\u003cb\u003eFig.\u0026nbsp;2B)\u003c/b\u003e. A comparison of survival curves of early health phase and transition phase showed a significant difference in response among all the tested concentrations (Log-rank [Mantel-Cox] Test, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003eFrom this study, we carefully choose 100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1000 \u0026micro;M concentrations of CUR to understand the DA-ergic neuroprotective efficacy of CUR in the ROT-mediated life phase-specific fly PD model. All these concentrations of CUR did not affect the survival of the fly at the selected treatment windows. Care was also taken in such a way that selected CUR concentrations do not cause any mobility defects. Results of the negative geotaxis assay (NGA) illustrated that the selected CUR concentrations induced no mobility defects in the fly of the early health phase and transition phase (\u003cb\u003eSuppl. Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 CUR rescues mobility defects mediated by ROT in both co- and pre-feeding regimens during the early health phase of\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe assessed the negative geotaxis ability of the ROT-mediated adult early health phase fly PD model to decipher the mobility alterations under both the CUR pre- and co-feeding conditions. The distance that the fly climbed in 12 sec was assayed after 4 days and 5 days of exposure to 500 \u0026micro;M ROT or ROT along with CUR (100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1000 \u0026micro;M). After 4 days of feeding the fly with ROT, it exhibited slowness in movement as indicated by a significant decrease in the speed (bradykinesia), which is the characteristic clinical feature of PD in humans. The speed of the fly was significantly improved when ROT was fed along with CUR (co-feeding regime) as compared to the fly fed with ROT alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The mobility of ROT-fed fly was significantly reduced after 5 days whereas the CUR co-feeding rescues the phenotype as observed through the improved speed of the fly, suggesting the protective efficacy of curcumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Pre-feeding the fly with CUR for 2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC \u003cb\u003e\u0026amp; D\u003c/b\u003e), 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eE \u003cb\u003e\u0026amp; F\u003c/b\u003e) and 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eG \u003cb\u003e\u0026amp; H\u003c/b\u003e) and exposed to ROT for 4 days and 5 days also showed similar rescue (significant protective effect at lower concentrations i.e. 100 and 250 \u0026micro;M indicates the resilience provided by CUR pre-feeding comparing to the co-feeding regimen), suggesting that observed protective efficacy of CUR is not through physical interaction and sequestration of the toxin. CUR \u003cem\u003eper se\u003c/em\u003e has no adverse influence on the mobility performance of the flies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 CUR fails to rescue mobility defects mediated by ROT in both co- and pre-feeding regimens during the late health and the transition phases of\u003c/b\u003e \u003cb\u003eDrosophila\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe adult life span of \u003cem\u003eDrosophila\u003c/em\u003e is categorized into a health phase, transition phase and senescent phase [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been shown that there is a significant variation (about 23%) in genome-wide transcript profiles with age in \u003cem\u003eDrosophila\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The efficacy of genotropic drugs depends on the availability of the target molecules at that stage of the life cycle. Hence, there is a possibility that targets of genotropic compounds such as CUR may not be present in all life stages [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo understand this paradigm, we tested CUR efficacy in the age groups of the late health phase (30 days old fly) and transition phase (50 days old fly). CUR could not improve the mobility defects induced by ROT in both the co- and pre-feeding regimens during the late health phase (\u003cb\u003eSuppl. Fig. S2\u003c/b\u003e) and transition phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e) of \u003cem\u003eDrosophila\u003c/em\u003e as quantified with climbing ability. The distance that the fly climbed in 12 sec after one day and two days of exposure to 25 \u0026micro;M ROT or ROT along with CUR (100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1000 \u0026micro;M) was assayed. Feeding with ROT alone significantly impaired the mobility of the fly while feeding the fly with CUR (500 \u0026micro;M) \u003cem\u003eper se\u003c/em\u003e did not show any effect on mobility. The co-feeding of CUR could not improve the mobility defects induced by ROT in both the late health phase (\u003cb\u003eSuppl. Fig. S2\u003c/b\u003e) and transition phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003e\u0026amp; B).\u003c/b\u003e Pre-feeding the flies of the late health phase with CUR exposed to ROT for 1 day and 2 days also could not rescue the mobility defects during late health phase (\u003cb\u003eSuppl. Fig. S2)\u003c/b\u003e. Pre-feeding the flies with CUR for 2 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC \u003cb\u003e\u0026amp; D\u003c/b\u003e), 3 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eE \u003cb\u003e\u0026amp; F\u003c/b\u003e) and 5 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eG \u003cb\u003e\u0026amp; H\u003c/b\u003e) and exposed to ROT for 1 day and 2 days also showed no improvement in mobility during the transition phase of the fly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith far-reaching limitations of therapeutic efficacy in NDDs like PD, which shows an average onset at 50 years (according to \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ninds.nih.gov/disorders/parkinsons\u003c/span\u003e\u003cspan address=\"http://www.ninds.nih.gov/disorders/parkinsons\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e disease), CUR failed to rescue the mobility defects in both the pre- and co-feeding regimens during late health phase (\u003cb\u003eSuppl. Fig. S2\u003c/b\u003e) and transition phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e) as indicated by negative geotaxis assay. These results suggest the limitation of CUR as a therapeutic compound in late-onset NDDs such as PD. However, whether CUR can be a prophylactic agent is yet to be ascertained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 CUR rescues \u0026ldquo;neuronal dysfunction\u0026rdquo; during the early health phase but not during the transition phase\u003c/h2\u003e \u003cp\u003eIn the \u003cem\u003eDrosophila\u003c/em\u003e brain, the number of DAergic neurons (DNs) in different clusters viz. PAL, PPL1, PPL2, PPM1/2 and PPM3 are 5, 12, 6/7, 8/9 and 6 respectively (\u003cb\u003eSuppl. Fig. S3\u003c/b\u003e). The number of DNs was quantified by using fluorescently labelled secondary antibodies targeting against the primary antibody which is the rate-limiting enzyme in dopamine synthesis, Tyrosine hydroxylase (TH). The image of the \u003cem\u003eDrosophila\u003c/em\u003e brain during the early health phase and transition phase captured through a fluorescence microscope is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eA respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough there is a natural variation in the number of DNs of the same neuronal cluster, no significant difference is observed among the different treatment groups analyzed during the early health phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and transition phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe FI of the secondary antibodies targeting the primary antibody (anti- TH) is an indicator of the amount of TH synthesis. Hence, the diminished FI can be an indicator of the level of DA-ergic neurodegeneration. During the early health phase of the fly, the FI of the fly fed with 500 \u0026micro;M ROT for 5 days was significantly reduced by 42.36%, 42.55%, 42.31%, 45.11% and 44.89% in PAL, PPL1, PPL2, PPM1/2 and PPM3 respectively when compared to control as observed under the fluorescence microscope. Co-feeding the fly of the early health phase with CUR could significantly improve the FI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In the case of transition phase fly, feeding with ROT alone led to significant reduction in the FI by 55.20%, 57.83%, 55.70%, 47.49%, and 56.74% in PAL, PPL1, PPL2, PPM1/2 and PPM3 respectively after 48 hr. Co-feeding the fly of the transition phase with CUR could not improve the reduction in FI (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe cluster-wise analysis of DA-ergic neuronal FI showed significant rescue in neuronal dysfunction in all the clusters during the early health phase but not during the transition phase of life stages of \u003cem\u003eDrosophila\u003c/em\u003e. The total intensity sum of all the DNs in the fly brain was clubbed together for further analysis. Results reveal that ROT significantly downregulates the FI (~\u0026thinsp;50%) (Suggesting diminished levels of TH protein synthesis) which could be significantly altered upon co-feeding with CUR during early health phase of the fly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). But the significantly reduced FI (~\u0026thinsp;50%) of the DNs during the transition phase of the fly could not be improved upon co-feeding with CUR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results indicate that CUR can rescue the neurodegeneration induced by ROT only during the early health phase but not during the transition phase of the fly.\u003c/p\u003e \u003cp\u003eThe results from the present study support the findings of the negative geotaxis assay which indicated that CUR rescues mobility defects only during the early health phase but not during the transition phase. These results reveal that the synthesis of the TH for DA synthesis is downregulated as the FI of the neuron is directly proportional to the level of TH. Hence, the level of dopamine synthesis may be reduced in the brain. This is ascertained by quantifying the level of DA and its metabolites using HPLC-ECD.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Curcumin replenishes diminished DA and its metabolites (DOPAC and HVA) only during early health phase but not during transition phase and doesn\u0026rsquo;t influence DA turn over\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eReduced DA-ergic neuronal FI is directly proportional to the diminished TH synthesis. Quantification of FI revealed that the intensity was significantly reduced under ROT-mediated conditions revealing the neurodegeneration which could be improved upon co-feeding with CUR during the early health phase but not during the transition phase of the fly. Also, CUR failed to improve the mobility defects induced by ROT in the transition phase fly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. Hence, we asked whether the failure in improving the climbing ability during this phase is due to CUR\u0026rsquo;s failure in replenishing brain dopamine levels. To confirm this correlation, we estimated the levels of DA and its metabolites (DOPAC and HVA) in fly brain tissue extracts of different treatment groups under study using HPLC-ECD (\u003cb\u003eSuppl. Fig. S4\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFeeding the fly with ROT alone led to a significant decrease in DA levels by 38% and 26% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), DOPAC levels by 38% and 13% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) and HVA levels by 29% and 21% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) during the early health phase and transition phase of the fly respectively. Co-feeding with 500 \u0026micro;M CUR and 1000 \u0026micro;M CUR could significantly increase the DA level by 17%, DOPAC levels by 21% and 18% and HVA levels by 12% and 14% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B,C) as compared to ROT-mediated group whereas lowest concentration of 250 \u0026micro;M CUR showed no significant difference in DA and HVA levels but improved the DOPAC level by 13% during the early health phase of the fly. Co-feeding the transition phase fly with CUR showed no improvement in the brain DA, DOPAC and HVA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003eA,B,C) as compared to ROT-induced group suggesting that efficacy of CUR is life stage-specific in fly PD model.\u003c/p\u003e \u003cp\u003eThis result indicates that CUR can rescue DA, DOPAC and HVA levels thereby rescuing the neurodegeneration during the early health phase but fails during transition phase of the fly. The present study suggests that CUR-mediated modulation of perturbations in DA metabolism is restricted to the adult early health phase and not to the transition phase. This illustration suggests that the genetic targets and molecular networks of genotropic drug CUR-mediated correction process may not be active/expressive at optimum levels during later phases of the adult life. From this result, it can be hypothesized that CUR rescues the perturbations in brain DA metabolism in ALSS fashion. This critical observation underlines the limitations of CUR as a therapeutic agent in the late-onset NDDs such as PD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe quantification of DA and its metabolites during the early health phase of the fly upon treatment with 500 \u0026micro;M ROT leads to a significant decrease in brain-specific DA, DOPAC and HVA when compared with control. Since mitochondria are involved in several metabolic pathways, it is possible that the inhibition of mitochondrial complex I by ROT also affects the metabolism of DA and downstream catecholamines. In the case of the transition phase fly, the level of DA, DOPAC and HVA is significantly lowered under ROT-mediated conditions. Upon co-feeding with CUR, the level of DA, DOPAC and HVA could be significantly rescued during the early health phase but not during the transition phase of the fly. This could be due to the lack of targets of CUR during the transition phase of the fly. Upon comparing the rescue patterns with respect to control, results reveal that there is no significant difference in DA turn over in both the health and transition phase of the fly. However, trend shows a marginal slow-down in the catabolism of DOPAC to HVA during the transition phase of the fly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn most populations, 3\u0026ndash;5% of PD is explained by genetic causes linked to known PD genes. However, the majority of patients (90\u0026ndash;95%) have idiopathic or sporadic PD [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Further, epidemiological studies indicate a relationship between various environmental factors such as pesticide exposure and the development of sporadic PD in later stages of life [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Hence, we developed an adult life stage (health and transition phase) specific fly model of PD using mitochondrial complex I inhibitor neurotoxicant rotenone [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Adult life phase specific gene expression profile variation across model organisms suggest the relevance and importance of life phase-specific animal models for understanding the pathophysiology of late-onset NDDs such as PD and screening small molecules/nutraceuticals with potential neuroprotective efficacy and deciphering molecular basis of their biological activity.\u003c/p\u003e \u003cp\u003eStudies on animal models have shown the neuroprotective efficacy of CUR [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Contradictory reports of concentration-dependent CUR toxicity have been reported in animal models [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and in \u003cem\u003eDrosophila\u003c/em\u003e model [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, the toxicity of CUR in animal models is under-reported. Hence, we performed a comprehensive experiment with an array of CUR concentrations to identify the non-toxic concentrations of CUR in both the adult early health phase and transition phase of the \u003cem\u003eDrosophila\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePresent experimental approach employing CUR was to expose the \u003cem\u003eDrosophila\u003c/em\u003e to a range of concentrations to determine its detrimental effects and select a suitable range of non-toxic concentrations for further studies. We employed 8 concentrations ranging from 25 \u0026micro;M to 2.5 mM of CUR in both the adult early health phase and transition phase male \u003cem\u003eDrosophila\u003c/em\u003e. It was found that CUR concentrations of 2.5 mM had a deleterious effect on the viability of the fly, whereas concentrations lower than 2.5 mM showed no observable toxicity in the early adult health phase fly up to 10 days (\u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e). In the highest concentration of 2.5 mM, all the flies employed in the experiment were dead by the 30th day and 11th day of CUR treatment in the case of the early health and transition phases respectively. The concentration below 1 mM did not show any mortality up to 10 days of exposure to CUR, indicating that there was no toxicity in all the lower concentrations. CUR toxicity study in human also suggests that it primarily causes gastrointestinal disturbances, diarrhoea, as well as distension and gastroesophageal reflux disease [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, it is essential to make out the toxic concentrations of CUR before employing it as a neuroprotective agent in the fly model of PD. Hence, we picked up sub-lethal concentrations of CUR (100 \u0026micro;M, 250 \u0026micro;M, 500 \u0026micro;M and 1 mM) and the same concentrations were employed in life phase-specific ROT-mediated \u003cem\u003eDrosophila\u003c/em\u003e model of PD under pre- and co-feeding regimen.\u003c/p\u003e \u003cp\u003eTo understand DA-ergic neurodegeneration and to decipher the effectiveness of therapeutic molecules, many laboratories either co-treat or pre-treat young PD fly models belonging to the health phase to screen small molecules/drugs/nutraceuticals and concluded about their DA-ergic neuroprotective efficiency [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. They determined the neuroprotective efficacy of the molecules by assessing behavioral markers such as mobility defects, biochemical markers such as anti-oxidant enzyme levels and levels of brain DA and its metabolite levels, or cytological markers such as degeneration of DNs in the whole brain of young animals whereas late-onset NDDs such as PD onsets during the transition phase of adult life!!\u003c/p\u003e \u003cp\u003eThe present study indicates that feeding the fly of the adult early health phase with ROT alone adversely decreases mobility (~\u0026thinsp;30%) which could be improved upon co-feeding with CUR. Feeding the fly with CUR \u003cem\u003eper se\u003c/em\u003e did not show any mobility defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003e\u0026amp; B\u003c/b\u003e). Results indicate that in both the co-feeding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA \u003cb\u003e\u0026amp; B\u003c/b\u003e) and pre-feeding regimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-H), CUR could confer neuroprotection during the early health phase. It has been reported that exposure to ROT induces severe mobility defects in \u003cem\u003eDrosophila\u003c/em\u003e and the same can be rescued upon co-feeding with CUR [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the case of the late health phase fly, CUR fails to rescue the mobility defects in both the co- and pre-feeding regimens (\u003cb\u003eSuppl\u003c/b\u003e. \u003cb\u003eFig. S2\u003c/b\u003e). In the case of the transition phase fly, CUR fails to rescue ROT-mediated mobility defects in both the co- and pre-feeding regimens as determined through the negative geotaxis assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results are in agreement with previous work from our lab, where CUR confers DA-ergic neuroprotection only during the early health phase but not during the transition stage [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. However, in the present study, CUR fails even during the late health phase, which can be attributed to mitochondrial complex I inhibitor ROT specific neurotoxicity. Previous studies demonstrated the neuroprotective efficacy of CUR in ROT-mediated neurotoxicity in cell culture, young adult \u003cem\u003eDrosophila\u003c/em\u003e and rat models and substantiated that CUR sequesters the intracellular and mitochondrial ROS levels and inhibits the caspase-3/caspase-9 activity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These results suggest that CUR may be a potential therapeutic compound for PD intervention. However, all these studies used young animals belonging to the adult early health stage. The results from the present study suggest that CUR can rescue ROT-mediated mobility defects in ALSS fashion; meaning CUR confers neuroprotection only during the early health phase and not in the late health phase and transition phase of the adult life.\u003c/p\u003e \u003cp\u003eThe death of DNs is the characteristic pathological marker of PD. Hence, in order to decipher the extent of DA-ergic neurodegeneration under induced PD conditions and possible rescue by CUR, we quantified the DNs in the whole fly brain followed by characterization of DA \u0026ldquo;neuronal dysfunction\u0026rdquo; by quantifying the FI emanated from the fluorescently labelled secondary antibody targeted against the primary anti-TH antibody using fluorescence microscopy. The results illustrate that there is no variation in the number of DNs between the control and PD brains of \u003cem\u003eDrosophila\u003c/em\u003e in both the health and the transition phases of adult life (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). This observation is consistent with the previous works from our lab [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and other laboratories [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], which is logical in the light of \u0026ldquo;dying back\u0026rdquo; phenomenon. The issue of loss of DNs \u003cem\u003eper se\u003c/em\u003e in fly brain has been thoroughly evaluated in multiple fly models of PD (both genetic and sporadic) and demonstrated that there is no structural loss of DA neurons, but only the synthesis of TH is diminished [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Hence, we went ahead with the quantification of the FI of TH-positive neurons. Results illustrate that the fly treated with ROT alone led to a significant decrease in FI to ~\u0026thinsp;50% which could be significantly rescued by 25\u0026ndash;30% upon co-feeding with CUR during the early health phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) while the diminished FI could not be rescued by CUR during the transition phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). We also analyzed the results by quantifying the FI of all the DNs present in the fly brain of different treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Similar results were obtained when analyzed in treatment group-wise. As the FI of the secondary antibodies is directly proportional to the level of synthesis of the TH, diminished levels of FI suggest the decreased levels of TH. Hence, it is possible that the level of DA synthesis might be reduced in the fly brain. This approach allowed us to accurately assess the extent of neurodegeneration of DNs in the fly PD models. This result is further substantiated by quantifying the levels of the brain-specific neurotransmitter DA and its metabolites (DOPAC and HVA) using HPLC-ECD.\u003c/p\u003e \u003cp\u003eThe present study shows that feeding the fly with ROT alone led to a significant reduction in the levels of DA, DOPAC and HVA during the early health and the transition phases of \u003cem\u003eDrosophila\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Similar results showing significant reduction in DA, DOPAC and HVA levels are indicated in 10 days old young fly [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], young mice [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], young wistar rat [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. All these results indicate that ROT significantly downregulates the DA level under induced PD conditions.\u003c/p\u003e \u003cp\u003eThe present study demonstrates that diminished levels of DA, DOPAC and HVA upon ROT treatment could be significantly rescued through co-feeding with CUR during the early health phase but not during the transition phase of the adult fly (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Study on young 12 weeks old albino wistar rat showed a significant reduction in DA and DOPAC levels upon ROT-mediated conditions which could be significantly improved upon co-feeding with CUR [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Another study indicated that feeding Danshensu (a plant extract; 60mk/kg for 28 days) could significantly improve the levels of DA, DOPAC and HVA in ROT-induced young 12 weeks old C57BL/6 mice indicating that the levels of DOPAC and HVA are proportional to DA content [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. CUR is shown to restore the levels of DA, DOPAC and HVA in neurodegeneration mediated by lipopolysaccharide in young Sprague-Dawley rats [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. All these results support the present study and indicate that CUR restores the levels of DA, DOPAC and HVA in ROT-mediated PD models. However, these studies are restricted to 5\u0026ndash;20 days young fly [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and 10\u0026ndash;12 weeks young mice and rat models [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] whose age is comparable with the early health phase of adult life. There is no data available relating to the levels of DA and metabolites in PD models of the transition phase of adult life, during which PD sets in. This is the first detailed study to have shown the levels of DA and its metabolites in the transition phase of \u003cem\u003eDrosophila\u003c/em\u003e model of ROT-mediated PD.\u003c/p\u003e \u003cp\u003ePresent study reveals that CUR does not influence the DA turn over during both the health and the transition phases of adult life. Research on DA metabolism gives an insight into the biochemical changes in DA-ergic system associated with PD. However, the biological significance of \u0026ldquo;turnover ratio\u0026rdquo; which denotes the level of metabolites (DOPAC and HVA) with respect to DA is not discussed in many studies quantifying DA and metabolites. Pagan et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] reported DA metabolites as \u0026ldquo;exploratory biomarkers\u0026rdquo; with reference to CSF levels of DOPAC and HVA. Although, compiling the CSF levels of HVA with xanthine improves its use as a biomarker; CSF levels of HVA poorly correlate with PD severity or progression [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Though DOPAC is the primary metabolite of DA [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], LeWitt [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] commented that representing the CSF level of DOPAC as a marker for the progression of PD is not valid though it shows severity. A significant amount of DOPAC and HVA, albeit lower DA levels, is seen in the human brain wherein DA-ergic innervation has not been implicated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Furthermore, the levels of neurotransmitters in CSF partially implicate the metabolism indicating limited relevance to neurotransmission in the brain. Hence, quantitative analysis of brain-specific DA and its metabolites concentrations are unrelated to DA-ergic neurotransmission which could be due to the possible role of capillary walls and glial cells in the catabolism of DA that needs to be further investigated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Hence, the notion \u0026ldquo;turnover\u0026rdquo; is not applicable with respect to the activity of DA-ergic system [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings suggest that CUR-mediated nourishment of DA metabolism is restricted to the adult health phase and not to the transition phase. This illustration indicates that genetic targets and molecular networks of genotropic drug CUR-mediated correction process may not be active/expressive at optimum levels during later phases of the adult life. However, there is no significant difference in the DA turn over pattern in both the health and transition phase of the fly but trend shows a marginal slow-down in the catabolism of DOPAC to HVA during the transition phase of the fly (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Thus the study underlines the limitation of CUR as a therapeutic agent in late-onset NDDs such as PD. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e9\u003c/span\u003e illustrates a detailed schematic presentation of our proposed study model.\u003c/p\u003e \u003cp\u003eThe present study provides insights into the underlying reasons for neuroprotective efficacy of CUR during the adult health phase of \u003cem\u003eDrosophila\u003c/em\u003e. Further, it also provides insights into the probable cause for the inefficacy of CUR during the adult transition phase of \u003cem\u003eDrosophila\u003c/em\u003e. It is important to figure out why CUR is failing to sustain the activity of the TH during the transition phase, understanding of which will have critical implications in developing therapeutic strategies for late-onset neurodegenerative diseases such as PD. Figuring out the underlying molecular networks in an ALSS fashion will be a path-breaking contribution in addressing the pathophysiology of PD.\u003c/p\u003e \u003cp\u003eThis is the first report to decipher the fact that CUR has limitations as a therapeutic molecule in the ROT-mediated idiopathic \u003cem\u003eDrosophila\u003c/em\u003e model of PD, and its efficacy is restricted only to the early health phase of adult life.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThe present study reveals that CUR mitigates the mobility defects induced by ROT during the early health phase but fails during the late health and transition phase in both the co- and pre-feeding regimens in the adult \u003cem\u003eDrosophila\u003c/em\u003e model of sporadic PD. Analysis of DNs reveals that their number remains unchanged under ROT-induced PD conditions but the FI of the secondary antibody targeted against the primary anti-TH antibody is significantly reduced suggesting the diminished levels of TH synthesis. The reduction in the FI could be improved upon co-feeding with CUR only during adult early health phase but not during the transition phase of the fly. Further analysis of the brain-specific DA and its metabolites reveals that CUR rescues DA and its metabolites during the early health phase only. The present study explains that curcumin\u0026rsquo;s ability to modulate perturbed DA metabolism is constrained to the adult early health phase. Observed contradictions can be attributed to the availability of genetic targets of genotropic nutraceutical curcumin in an adult life phase-specific fashion. Hence, it confers DA-ergic neuroprotection in adult early health phase but not in transition phase.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare that there is no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM. Ayajuddin: Data curation; Formal analysis; Investigation; Methodology; Software; Visualization; Roles/Writing - original draft; and Writing - review \u0026amp; editing. A. Das: Formal analysis; Methodology; Software; Visualization; Data curation; Writing - review \u0026amp; editing. S.C. Yenisetti: Conceptualization; Methodology; Funding acquisition; Project administration; Resources; Supervision; Validation; Visualization; Writing - review \u0026amp; editing; and Data curation.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research is supported by the Department of Biotechnology (DBT), India (R\u0026amp;D grant no. BT/405/NE/U-Excel/2013) awarded to SCY. Part of the work was presented at the International Conference on Recent Advances in Biotechnology and Environmental Science, organised by Vellore Institute of Technology (VIT), Vellore, India, 16-18 December, 2022; 4th International Conference on Neurology and Brain Disorders (INBC-2021), 9-10 September, 2021, Italy, Rome by MA (for which MA was awarded with international travel fellowship by IBRO (International Brain Research Organization, France); and at International Conference on Neuroscience and Neurological Disorders and 37th Annual Meeting of Society for Neurochemistry (SNCI), India, organised by Department of Biomedical Engineering, North Eastern Hill University, Shillong, Meghalaya, India. 14-16 September, 2023 by SY (plenary talk). MA received DBT (Department of Biotechnology, India)-JRF (junior research fellowship) and SRF (Senior research fellowship) from ICMR (Indian Council of Medical Research), India. AD received JRF and SRF from DBT.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSaleh, M.A., Amer-Sarsour, F., Berant, A., Pasmanik-Chor, M., Kobo, H., Sharabi, Y., et al. Chronic and acute exposure to rotenone reveals distinct Parkinson's disease-related phenotypes in human iPSC-derived peripheral neurons. Free Radic Biol Med. 213, 164\u0026ndash;173 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePouchieu, C., Piel, C., Carles, C., Gruber, A., Helmer, C., Tual, S., et al. Pesticide use in agriculture and Parkinson's disease in the AGRICAN cohort study. Int. J. 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GardeninA confers neuroprotection against environmental toxin in a \u003cem\u003eDrosophila\u003c/em\u003e model of Parkinson\u0026rsquo;s disease. Commun Biol. 4(1), 162 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePandareesh, M.D., Shrivash, M.K., Naveen Kumar, H.N., Misra, K., Srinivas Bharath, M.M. Curcumin monoglucoside shows improved bioavailability and mitigates rotenone induced neurotoxicity in cell and \u003cem\u003eDrosophila\u003c/em\u003e models of Parkinson's disease. Neurochem Res. 41(11), 3113\u0026ndash;3128 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePhom, L., Achumi, B., Alone, D.P., Muralidhara, Yenisetti, S.C. Curcumin's neuroprotective efficacy in \u003cem\u003eDrosophila\u003c/em\u003e model of idiopathic Parkinson's disease is phase specific: implication of its therapeutic effectiveness. 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Curcumin effectively rescued Parkinson's disease-Like phenotypes in a novel \u003cem\u003eDrosophila\u003c/em\u003e melanogaster model with dUCH knockdown. \u003cem\u003eOxid Med Cell Longev\u003c/em\u003e. 2018:2038267 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuratta, S., Chiaradia, E., Tognoloni, A., Gambelunghe, A., Meschini, C., Palmieri, L., et al. Effect of curcumin on protein damage induced by rotenone in dopaminergic PC12 Cells. Int J Mol Sci. 21(8), 2761 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNaser, A.F.A., Aziz, W.M., Ahmed, Y.R., Khalil, W.K.B., Hamed, M.A.A. Parkinsonism-like disease induced by rotenone in rats: Treatment role of curcumin, dopamine agonist and adenosine A2A receptor antagonist. 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JAMA Neurol. 77(8), 1038\u0026ndash;1039 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEbinger, G., Michotte, Y., Herregodts, P. The significance of homovanillic acid and 3,4-dihydroxyphenylacetic acid concentrations in human lumbar cerebrospinal fluid. J Neurochem. 48(6), 1725\u0026ndash;1729 (1987).\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, Rotenone, Transition stage","lastPublishedDoi":"10.21203/rs.3.rs-4645640/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4645640/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpidemiological studies suggest a strong linkage between exposure to environmental toxins and onset of Parkinson\u0026rsquo;s disease (PD). Rotenone is a widely used pesticide and known inhibitor of mitochondrial complex I, that has been shown to induce Parkinsonian phenotypes in various animal models. Our laboratory has developed a rotenone mediated ALSS \u003cem\u003eDrosophila\u003c/em\u003e model of PD which is critical to screen small molecules and identify molecular targets of dopaminergic neuroprotection for late-onset neurodegenerative diseases such as PD. Using negative geotaxis assay, qualitative and quantitative analysis of dopaminergic neurons by fluorescence microscopy and further quantifying the levels of dopamine and its metabolites by HPLC, we have assessed the neurodegeneration under PD induced conditions and neuroprotection by employing curcumin in \u003cem\u003eDrosophila\u003c/em\u003e model of PD. Exposure to rotenone induces mobility defects in health and transition phase of adult \u003cem\u003eDrosophila;\u003c/em\u003e whereas curcumin ameliorates the deficits only during early health phase but fail during late health and transition phases. Probing the whole fly brain using anti-tyrosine hydroxylase antibodies, for rotenone mediated dopamine neurodegeneration illustrates that it does not cause loss of dopaminergic neurons \u003cem\u003eper se\u003c/em\u003e. However, it leads to dopaminergic \u0026ldquo;neuronal dysfunction\u0026rdquo; (diminished levels of rate limiting enzyme of dopamine synthesis) and curcumin rescues the neuronal dysfunction only during the early health phase but fails to mitigate the dopamine neuronal pathology during the transition phase of adult life. Genotropic nutraceutical curcumin replenishes the diminished levels of brain specific dopamine and its metabolites DOPAC and HVA during adult early health phase and fails to do so in adult transition phase, suggesting that the life phase-specific dopaminergic neuroprotective efficacy is mediated through differential modulation of perturbations in brain dopamine metabolism. Present study suggests the limitation of curcumin as a therapeutic agent for PD and emphasizes the necessity of screening putative neuroprotective small molecules for late onset neurodegenerative diseases such as PD in life phase matched animal models during which the disease sets in.\u003c/p\u003e","manuscriptTitle":"Age specific neuroprotection of curcumin is through differential modulation of brain dopamine metabolism: Insights from Drosophila model of Parkinson’s disease ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-27 18:05:25","doi":"10.21203/rs.3.rs-4645640/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":"0b30f8c6-2e3b-400c-ab40-173122b3d02a","owner":[],"postedDate":"July 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":34891689,"name":"Biological sciences/Cell biology"},{"id":34891690,"name":"Biological sciences/Neuroscience"},{"id":34891691,"name":"Biological sciences/Zoology"}],"tags":[],"updatedAt":"2024-09-12T08:41:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-27 18:05:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4645640","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4645640","identity":"rs-4645640","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}