Differential regulation of brain-specific molecular pathways is the reason for curcumin’s adult life-phase specific DAergic neuroprotection: Insights from ALSS Drosophila model of Parkinson’s disease

(150-250 words) Curcumin (CU) , a bioactive compound of turmeric, has been put forward as a “golden molecule” due to its anti -inflammatory, antioxidant, hepatoprotective, neuroprotective, and anti-cancer ability, as proven by research conducted over the decade and more. Our laboratory, developed an adult life stage specific (ALSS) Drosophila model of sporadic Parkinson’s disease (PD), and for first time demonstrated that dopaminergic (DAergic) neuroprotective efficacy of curcumin is limited to health phase viz. adult-young life stage and is absent during transition phase viz. adult senior life stages when PD set in. This observation suggests the limitation of curcumin as a therapeutic agent for late-onset disorders like PD. Further, our laboratory also demonstrated that despite curcumin’s ability to sequester oxidative stress during both the adult life stages, neuroprotection and brain dopamine replenishment is granted only in health stages but not in a vulnerable transition stage, which prompted to put forward the hypothesis that the molecular target(s) of CU, may be absent or inadequate in the transition stage of aging brain. With this insight, the current study was implemented to analyse the life stage-specific differential regulation of multiple molecular players of neuro-integral pathways in brain of ALSS Drosophila model of PD with curcumin intervention. It is discovered that curcumin-mediated health phase-specific neuroprotection underlies the correction of an altered expression of 1. dFOXO, GADD45, Puc of Bsk-dFOXO stress response pathway, 2. Mfn2 of Mitochondrial dynamics 3. CncC, GCLC, Prx 2540 -1,2, Jafrac1, Prx3 of Phase II antioxidant defense system pathway. Further, it is discovered that significant aging-associated naturally altered expression of certain molecular targets exists , that may contribute to the limitation of curcumin’s DAergic neuroprotective efficacy during the adult-transition stage. This knowledge will help in developing altered therapeutic strategies for PD as molecular targets of curcumin are conserved among fly, mice and human. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 3

Curcumin, health phase, transition phase, Parkinson’s disease, neuroprotection, molecular targets Abbreviation: CU: Curcumin DAergic: Dopaminergic HP: Health Phase SNpc: Substantia Nigra pars compacta TP: Transition Phase DA: Dopamine PD: Parkinson’s Disease L-DOPA: Levodopa NDD: Neurodegenerative diseases ALSS: Adult life stage specific 1. Introduction The complicated interplay of gene and environment superimposed on slow and sustained neuronal dysfunction associated with aging [1,2] underlies cases of sporadic Parkinson’s disease ( PD) in the population over the age of 60 [3]. The physiological symptoms are associated with motor and non-motor symptoms due to the death of dopaminergic (DAergic) neurons in the SNpc region of the human brain leading to the depletion of dopamine ( DA) level [4]. Modern drug therapy of PD focuses on addressing the symptoms and consists mainly of d opamine (DA) supplementation through DA agonists like L-DOPA (Levodopa) or DA preservation through Catechol-o-methyltransferase and L-Monoamine Oxidase inhibitors [5]. As a part of combination therapy, L-DOPA-mediated DA supplementation is established as the gold standard treatment for PD and is the only therapeutic measure to counter late- onset symptoms of the dis ease [6]. However, L-DOPA-mediated therapy has been documented to be uncertain as it goes through “On -Off” phases, i.e., during the “On” phase patients respond to the therapy and during the “Off” phase patients do not respond to the therapy [7]. Further prolonged treatment with L-DOPA leads to the manifestatio n of “DA - resistant” motor , non-motor symptoms and neurotoxicity [8]. Therefore, symptomatic preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 4 treatments are not enough to combat the onset and progression of PD. Hence, disease- modifying treatments such as nanoformulations, small molecules, immunotherapies, etc, are proposed as the new “cutting-edge tools” that may alter PD etiopathogenesis at the root level [6]. Multi-acting nutraceutical CU has gained potent interest in combating various chronic diseases including PD due to its genotropic property, i.e. ability to act on multiple molecular players [9]. In recent years, curcumin ( CU) has also undergone human trials for PD. The efficacy of CU in human PD condition was also tested in a recent study. It was found that nano micelle CU treatment (80 mg/da y for 9 months) on idiopathic PD patients (≥30 years of age) demonstrated significant improvement in a few aspects, overall, this did not culminate into statistically significant results for the whole trial [10]. The author concluded that although CU is a safe natural substance, this trial could not demonstrate its effectiveness in improving PD patients' clinical symptoms and quality of life [10]. Similarly, CU’s limitation has been reflected in multiple chronic diseases in clinical trials [9]. Therefore, the inference is that the efficacy of CU in pre-clinical trials could not be translated to the human condition, and the current consensus is that future trials of any such nutraceuticals may directly be done in humans to cut short the discovery time and research expenditure [9]. However, it is important to note that in-vitro cell model studies might not recapitulate the in-vivo disease etiopathogenesis. Further, insights from the works in our laboratory suggest that when modeling a disease in vivo, variables such as life stages and their physiological implications on disease progression must be considered, so that the onset and progression of a disease match as near as possible to the human condition [11, 12]. Therefore, employing young animal models that do not match with appropriate life stages of disease onset in humans can be a possible reason for the failure to translate the results of preclinical studies to clinical set- up. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 5 With this insight, our lab focused on deciphering the neuroprotective efficacy of CU in an ALSS Drosophila model of sporadic PD developed in-house. The adult life of Drosophila is characterized by three stages, viz. health phase (HP) (No apparent mortality) corresponding to the adult young life stage, transition phase (TP) (10% mortality) corresponding to middle age of adult life and senescence phase (Steady decline in survival) corresponding to old age of adult life [13]. The aging of Drosophila is associated with alteration in genome-wide transcription profile by 23% [14]. Changes in genome-wide transcription profile during different life phases of Drosophila are similar to humans where significant aging-associated gene expression changes contribute to potent risk factors for an array of disorders like PD [15, 16]. Keeping the critical aspect that aging is a potential risk factor for late-onset NDD like PD, previously our laboratory developed the ALSS fly model and demonstrated that CU’s DAergic neuroprotective efficacy is HP-specific [12]. The same ALSS PD fly model is employed in the present study with an aim to understand the pathophysiology associated with PD before the occurrence of organismal death (employed neurotoxicant concentrations cause no mortality at the time points where phenotypes were scored) and to decipher the molecular basis of adult life phase specific CU’s DAergic neuroprotective efficacy. To understand the implication of ALSS neuroprotective efficacy of CU in the PD model at neuronal and neurochemical levels, in-situ DAergic neurons and ex-vivo DA and metabolites were quantified with fluorescence microscopy and HPLC-ECD method. Our laboratory demonstrated that the onset of sporadic PD does not lead to degeneration of DAergic neuronal cell bodies but rather induces DAergic “neuronal dysfunction” during both HP and TP [under review]. “Neuronal dysfunction” is termed as the reduction of synthesis of tyrosine hydroxylase (TH), which is a rate-limiting enzyme for the synthesis of DA [17]. Further, diminished TH synthesis resulted in diminished DA level and the PD condition also prompted preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 6 enhanced DA turnover, which may contribute to more summative oxidative stress [under review]. On the other hand, OS markers have been considered as the gold standard in the analyses of the neuroprotective efficacy of nutraceuticals since it is viewed as a contributing factor to NDD like PD [18-22]. In the PQ -induced ALSS (health and transition phas e) PD fly model, Phom [23] quantified the oxidative stress (OS) in brain using OS markers viz., reactive oxygen species (ROS) level, protein carbonyl (PC) level, malondialdehyde (MDA) level, hydroperoxide (HPer) level, catalase (CAT) activity, superoxide d ismutase (SOD) activity, glutathione s transferase (GST) activity, GSH level and neurotransmitter acetyl-cholinesterase (AChE) activity. It was demonstrated during both the adult life phases that levels of brain ROS, PC, HPer MDA and activity of CAT, SOD, GST were enhanced in induced PD condition. Further, exposure to PQ also showed downregulation of brain GSH level and AChE activity. However, CU intervention sequestered the upregulated ROS, PC, MDA, HPer levels, inhibited enhanced SOD, CAT, GST activity an d rescued diminished GSH level, AChE activity [23]. It is important to note that though the brain OS is sequestered during both phases of adult life, CU fails to confer DAergic neuroprotection during TP, as is evident from its failure to rescue the brain DA level [12]. This insightful study illustrates that sequestration of oxidative stress may be necessary but is not sufficient to confer DAergic neuroprotection in PD. It is argued that apart from oxidative stress, other molecular players/pathways may hav e synergic effects that could be responsible for the observed DAergic neurodegeneration/neuroprotection [12]. Further, this evidence emphasizes the importance of employing life -stage matched animal models for late -onset NDD such as PD and stresses the limi tation of OS and inflammation markers perse -based studies to determine DAergic neuroprotective efficacy of nutraceuticals/therapeutic agents/small molecules. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 7 Herbicide paraquat (PQ) is a multi-hit environmental neurotoxicant whose underlying mechanism of dopaminergic (DAergic) neurodegeneration may underlie ROS generation, deficiency in antioxidant enzyme levels, neuroinflammation, mitochondrial dysfunction, and ER stress, leading to a cascade of molecular crosstalks that result in the initiation of apoptosis [24]. Therefore, to understand the mechanism of DAergic neuroprotective efficacy of CU, it is necessary to investigate the genetic/molecular players and their possible age-mediated differential regulation. It has been demonstrated that multiple molecular pathways are involved in the DAergic neuronal integrity. JNK/BSK signalling pathway and its downstream targets through FOXO/dFOXO axis is known for adaptive stress response and when regulated rightly can promote life span extension, neuroprotection [25 - 29]. IIS- mTOR/dTOR pathway is known for actively controlling anabolic processes and metabolism, impairment of such metabolic process known as “metabolic consequences” is a common phenomenon in onset and progress of PD [30-32]. The adaptive stress response controlled by JNK/Bsk signalling pathway and anabolic process controlled by IIS- mTOR/dTOR pathway influences mitochondrial dynamics which may also contribute to progression of NDD like PD [33 -35]. Further, phase II ADS producing/utilizing GSH for antioxidant response [36, 37] and metal homeostasis pathway which prevents free metal accumulation in neuron [38, 39] are potent biological pathways that are involved in neuroprotection/neurodegeneration. Hence understanding the possible differential modulation of molecular players involved in these signalling networks may throw an opportunity to understand the ALSS neuroprotective efficacy of CU, understanding of which will be of great importance and support to develop novel treatment methods/regimen and alter existing therapeutic strategies preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 8 2. Materials and Methods 2.1. Modelling ALSS-PD in Drosophila and therapeutic intervention Generation of the PQ-mediated early onset, late-onset sporadic PD model of Drosophila and therapeutic intervention with CU co-feeding is described in Phom et al [12]. The study of Phom et al [12] highlights the ALSS neuroprotective efficacy of CU. The current study uses the same model, where detailed description and results relating to selection of neurotoxicant and CU concentrations were provided [12]. 2.1.1. Drosophila stock and husbandry Oregon K (OK) flies used in this study were obtained from the National Drosophila Stock Centre, University of Mysore, Mysore, Karnataka, India. Flies were maintained in food media containing (Sucrose, Agar-Agar, Yeast, and Propionic acid) [12, 40], under standard laboratory conditions of 22±2 oC temperature with humidity of 60%, and 12:12 hrs light and dark condition. 2.1.2. Collection and aging of adult male files For the collection of adult male flies, the parental generation was transferred to a fresh media vial for laying eggs for 4 days and then removed. After 11-12 days the eclosed flies were lightly anaesthetised to separate males and females. Male flies were collected in food media vials (25 male flies in each vial) and aging was done according to the necessity of the experiment while switching them into fresh media vials every 4 th day. Flies belonging 4-5 days and 50-55 days representing HP and TP of adult life span respectively were used to model early and late-onset forms of PD as described in Phom et al [12]. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 9 2.1.3. Chemicals for feeding and exposure Sucrose (Cat. No. 1947139) procured from Sisco Research Laboratory (SRL, Mumbai, India), Type I Agar Agar (Cat. No. GRM666) procured from HiMedia (Thane, India), Propionic acid (Cat. No. 8006050-500 -1730) procured from MERCK (Rahway, USA) and market-available sugar tolerant dry yeast (Angel, instant dry yeast) were used for food media preparation. For the exposure regimen, Methyl viologen dichloride hydrate /Paraquat (PQ) (Cat. No. 856177), CU (Cat. No.C1386) and Dimethyl Sulphoxide (DMSO) (Cat. No. D8418) were purchased from Sigma Aldrich (St. Louis, MO, USA). Feeding on Whatman filter paper no.1 disc in a 30x100 mm glass vial was preferred as the exposure methodology for the experiment. 2.1.4. PQ treatment and CU co-treatment protocol The necessary amount of PQ was dissolved in 5% sucrose solution to prepare 10 mM of PQ solution. A primary stock of 200 mg CU in 1 ml of DMSO was prepared (543 mM). Co-feeding of CU with PQ was achieved by dissolving 1.38 µL and 2.76 µL of CU stock in 1.5 ml of 10 mM PQ to obtain 500 µM and 1 mM of CU concentrations respectively. Also, exposure to CU per se at the concentrations (500 µM and 1 mM) was achieved by dissolving 1.38 µL and 2.76 µL of CU stock in a 5% sucrose solution only. Flies of the control group were fed 275 µL of 5% sucrose on filter discs while flies of experimental groups were fed 275 µL of 10 mM PQ (induced PD group), CU 500µM/1mM + 10 mM PQ (CU co-treatment group) and CU 500µM/1mM + 5% sucrose (CU per se group) respectively on filter discs. With 25 flies per vial, a minimum of 50-100 flies were exposed per group for 24 hrs [12]. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 10 2.2. Extraction of fly brain RNA and performing quantitative real time PCR (qRT- PCR) to quantify gene expression profiles: The methodologies pertaining to gene expression analysis utilizing qRT-PCR is partially described in Das et al [41]. 2.2.1. Chemicals and materials for gene expression analysis RNaseZAP (Ambion, Waltham, USA, Cat: AM9780), DEPC treated water (HiMedia, Thane, India, Cat: ML024), TRIzol reagent (Invitrogen, Waltham, USA, Cat:15596026), Chloroform (Sigma-Aldrich, St. Louis, USA, Cat: c2432), Isopropyl alcohol (HiMedia, Thane, India, Cat: MB063 -1l), DEPC treated water (HiMedia, Thane, India, Cat: ML024), Ethanol (MERK, Rahway, USA, Cat: 1. 00983.0511), DNase (Invitrogen, Waltham, USA, Cat:18068-015), Oligo(dT) (Invitrogen, Waltham, USA, Cat: 18418012), dNTP mix (Qiagen, Hilden, Germany Cat: 14505289), SuperscriptTM II reverse transcriptase (Invitrogen, Waltham, USA, Cat: 18064014), PowerUp™ SYBR® Green Master Mix (Applied Biosystems, Waltham, USA, Cat: A25742), 96 reactions well plate (Biorad, Hercules, USA, Cat: MLL-9601). 2.2.2. Total mRNA isolation from fly head In brief, post-exposure each group of flies were frozen and head s were decapitated. 50 heads per fly group w ere used to extract total RNA extraction using Guanidium thiocyanate phenol chloroform extraction or the TRIzol method. Tissues were homogenized with 1 mL of TRIzol and centrifuged (At 12g for 15 mins at 2˚C) to discard the tissue debris. Supernatant was collected and incubated at room temperature preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 11 for 5 mins. Phase separation by chloroform ( 200 µL was added and vigorously shaken followed by incubation at room temperature for 2 -3 mins) was opted to be the best way to iso late aqueous solution containing RNA from the homogenate. The tube s are centrifuged at 12g for 15 minutes at 2˚C . Colourless upper aqueous phase was collected and RNA was eluted and precipitated out from the aqueous solution by adding chilled Isopropyl alcohol (500 µL was added and incubated at room temperature for 10 minutes followed by centrifugation at 13g for 15 min at 2˚C). The pellet of RNA was washed with 1 mL of 75% ethanol to remove salt content from TRIzol reagent. Pellet was air dried and resuspended in 30-35 µL DEPC treated water (Incubated for 10 minutes at 55˚ to 60 ˚C to dissolve the pellet). RNA was quantified and stored in -80oC freezer till further use. Precaution taken to avoid RN ase contamination, so every instrument and working space was cleaned with RNase ZAP prior to the experiment. 2.2.3. First strand cDNA synthesis RNA was quantified with pedestal method using Nanodrop (Thermo-Fisher scientific). 1 ug of RNA from each group is to be synthesized into single stranded cDNA. Required volume of RNA which contains 1 ug of the biomolecule was taken and treated with DN ase+DNase buffer (1 µL each) to avoid DNA contamination. Volume makeup for each sample was done by adding DEPC water to obtain a uniform volume of 10 µL. Sample was incubated 15 mins at room temperature . After which 1 µL of EDTA was added followed by heating at 65oC for 10 minutes to deactivate the DN ase. Tubes were puff centrifuged and Oligo DT and dNTP mix (1 µL each) were added followed by heating at 65oC for 5 minutes to promote annealing. Tubes were chilled immediately and then puff centrifuged. First strand buffer (4 µL) and 0.1 M DTT (2 µL) were added followed by heating at 42 oC for 2 minutes to prepare the RNA template for first strand cDNA synthesis. 1 µL of Superscript II reverse transcriptase was added and incubated at 42oC preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 12 for 50 minutes. The reaction was deactivated by incubating the tubes at 70 oC for 15 minutes. Tubes were puff centrifuged again and now the first strand cDNA is ready which is stored in -20oC freezer till further use. 2.2.4. Gene expression analysis The Real time PCR reaction was carried out in Applied Biosystem (ABI) Step-One Plus thermal cycler (Thermo fisher scientific), using Power Up SYBR green master mix. cDNA was diluted with DEPC water with a factor 5 for the reaction. Reaction volume was considered 10 µl which contains 1 µl of diluted cDNA, 8.6 µl of SYBR green and 0.2 µl of forward and reverse primer specific to the internal control RP49 and target gene dFOXO. Reaction plate was loaded accordingly as described in Das et al, 2021. The thermal cycler protocol for the qRT-PCR is as follows Amplification (Amp.) protocol: Holding stage @ 95oC C for 10 mins Amp. Cycle: (95oC for 15 secs followed by 60oC for 1 min) X 40 Melting Curve analysis: Quickly ramped up to 94 oC followed by cooling at 60oC The C T values were obtained and the relative fold change to the Control sample was measured by the 2-∆∆Ct method as described by [42]. For the experiment, in different treatment groups, utilizing the afore mentioned methodologies differential gene expression was analyzed for the molecular players belonging to JNK signaling pathway, IIS-mTOR signaling pathway, mitochondrial dynamics, Phase II antioxidant defense response and metal homeostasis (Table 1). Flybase Gene Gene name Oligos preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 13 ID CG7939 RP49 F 5’-AGGGTATCGACAACAGAGTG-3’ R 5’-CACCAGGAACTTCTTGAATC-3’ CG5680 Bsk F 5’-CACTCAGCAGGAATTATTCACAG-3’ R 5’-TTAGAGTGCAGTCGGCCTTT-3’ CG3143 dFOXO F 5’-TCGAGTGCAATGTCGAGGAG-3’ R 5’-AGCGGTATATTGATGTCCAGCAG-3’ CG4533 l(2)efl F 5’-CAGACGCGTTTATCCAAGTG-3’ R 5’-ATCCCACCAGTCACGGAAC-3’ CG8846 4e-bp F 5’-CCAGA TGCCCGAGGTGTA-3’ R 5’-AGCCCGCTCGTAGA TAAGTTT-3’ CG11086 GADD45 F 5’-GATCCCTCTTCTGCCTGATG-3’ R 5’-CAGCAGTACCTCGTGCATGT-3’ CG5436 HSP68 F 5’-GGAGGCTCCACTCGTATTCC-3’ R 5’-TCTTTCCGCCGAAGAAGTT-3’ CG7850 Puc F 5’-GCCACATCAGAACATCAAGC-3’ R 5’-CCGTTTTCCGTGCATCTT-3’ CG11793 SOD1 F 5’ -CAAGGGCACGGTTTTCTTC-3’ R 5’-CCTCACCGGAGACCTTCAC-3’ CG8905 SOD2 F 5’-AATTTCGCAAACTGCAAGC-3’ R 5’-TGATGCAGCTCCATGATCTC-3’ CG6871 Catalase F 5’-TGACTACAAAAACTCCCAAA-3’ R 5’-TTGATTCCAATGGGTGCTC-3’ CG8167 ILP2 F 5’-ATCCCGTGATTCCACACAAG-3’ R 5’-GCGGTTCCGATATCGAGTTA-3’ CG14167 ILP3 F 5’-CCGAAACTCTCTCCAAGCTC-3’ R 5’-GCCATCGATCTGATTGAAGTT-3’ CG33273 ILP5 F 5’-CAAACGAGGCACCTTGGG-3’ R 5’-AGCTATCCAAATCCGCCA-3’ preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 14 CG5686 Chico F 5’-GGCATACGGGCAGCTAGAC-3’ R 5’-TTCTTGAGGTAGCCACTCAGC-3’ CG5092 dTOR F 5’-GCTCAGAGGCGAGAGACAAG-3’ R 5’-CCAGCTCACGGAGGATAAAG-3’ CG10539 RS6K1 F 5’-TGACCTAGAACCGGAATTGTG-3’ R 5’-TCCTCGCAGAGCTGTATGG-3’ CG3114 Ewg F 5’-CTCCCAGTCGAGTGCTAACC-3’ R 5’-ACCGTTTGCGTGGTGTAATC-3’ CG4217 TFAM F 5’-CAAGTGGACTCGCCTTTCC-3’ R 5’-TCATCTTCTCCTCCCAAACG-3’ CG3869 Mfn2 F 5’-ACCTCACCTCGGCCAACT-3’ R 5’-GTGGTGGCGGTATCAACC-3’ CG6030 ATP SynD F 5’-TCAACAAGCCCACCTTCTG-3’ R 5’-TGCTCCTTGGACTTGTAGCC-3’ CG17894 CncC F 5’-TTCACAGATATCAACAGTGTCAT-3’ R 5’-CAGGGGCAAGCGTATGTATT-3’ CG2259 GCLC F 5’-CGAGGAGAATGAGCTGTTCC-3’ R 5’-ACCAGACCCGGAAAAACG-3’ CG4919 GCLM F 5’-GGCAATTGGCTATTGTGTGTC-3’ R 5’-GGGCTCAAATCGCTAAACC-3’ CG10045 GSTD1 F 5’-TCGCGAGTTTCACAACAGAA-3’ R 5’-TGAGCAGCTTCTTGTTCAGC-3’ CG12405, CG11765 Prx 2540-1,2 F 5’-GCAACGTTGACGAGATTCTG-3’ R 5’-GCAGGATCATGACCTTAGTGC-3’ CG1633 Jafrac1 F 5’-GCGGCCATTTTGTAGAGTTC-3’ R 5’-AGCGGGCTTCTGTAGCTG-3’ CG1274 Jafrac2 F 5’-ACTCTTCTACCCACTGGACTTCA-3’ R 5’-CTTCTTGAACTCGGCGATG-3’ CG7217 Prx5 F 5’-AAATTTCTTGGCCGAGTTGTTA-3’ preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 15 Table 1: Details of the gene specific primers for expression analysis The specificity of the primers were validated with melting curve analysis (Supplementary Information 2). 2.2.5. Statistical analysis Graphs were created using GraphPad Prism 8.4.3 software, statistical analysis was completed, and results were expressed as the mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) followed by Tukey post-hoc test and unpaired t-test was carried out to draw significance for the gene expression data. P-values under 0.05 were regarded as significant. 3.

3.1. CU-mediated ALSS modulation of JNK/Bsk Signalling pathway R 5’-GATGGCAGGGAGTCTCCTACT-3’ CG5826 Prx3 F 5’-GAAGACTACAGGGGCAAGTACC-3’ R 5’-GGGGCAAACGAATGTGAA-3’ CG9470 MtnA F 5’-AACTCAATCAAGATGCCTTGC-3’ R 5’-TTGCAGGATCCCTTGGTG-3’ CG2216 Fer1HCH F 5’-TGCTAGCCTGCTCCTGTTG-3’ R 5’-GTCCACCCAGTCCTTGGTAA-3’ CG4900 IRP-1A F 5’-CTCCA TCGACAGCAAATA TGAG-3’ R 5’-CCAGCACA TGAAAGTTGTCAC-3’ CG6342 IRP-1B F 5’-CGCCCAGTTCGAGAAAAC-3’ R 5’-GGATCGAGTAGGGCAGTTGA-3’ preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 16 3.1.1. CU fails to rescue diminished Bsk expression in the PD brain during HP and TP Bsk-signalling promote neuroprotection when activated in moderation (Gan et al., 2021). In the present study, Bsk expression in the PD brain was inhibited by 39% (P<0.01) during HP and by 51% (P<0.001) during TP. CU intervention did not alter the diminished expression of Bsk during both the adult life stages. However, feeding of CU per se upregulated Bsk expression by 87% (Compared to the control brain) (P<0.001) during HP but not during TP ( Fig 1a). The observation suggests that while CU can modulate Bsk expression during HP in the physiologic condition, its intervention has no influence in modulating Bsk in PD brain of both life phases. Further, we tried to investigate the aging- associated changes in Bsk expression in the healthy brain. It was observed that in the TP fly brain, Bsk expression is downregulated by 54% (P<0.001) as compared to that of the HP (Fig S1a). 3.1.2. dFOXO level was diminished in the PD brain and CU intervention rescues only during HP The downstream effect of Bsk signalling is brought about activation of transcription factor dFOXO, promoting gene expressions associated to adaptive stress response, neuroprotection, neuronal maintenance and longevity [25,43,44]. In the present study, dFOXO expression in the PD brain was inhibited by 44% (P<0.01) during HP and by 55% (P<0.01) during TP ( Fig 1 b). However, CU intervention (500 µM and 1 mM) rescued diminished dFOXO level significantly (P<0.05 and P<0.01) during HP but not during TP. CU per se feeding also upregulated dFOXO expression by 2 fold (compared to control brain) (P<0.001) during HP but not during TP ( Fig 1b). The observation suggests that dFOXO expression modulation in PD and physiologic conditions is a feature of CU preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 17 that is restricted only to HP. Hence, CU-mediated ALSS neuroprotection underlies the modulation of dFOXO of the Bsk signalling pathway. Further investigation revealed that in healthy TP brain, dFOXO expression is downregulated by 16% (P<0.01) as compared to that of HP (Fig S1b). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 18 Figure 1: CU-mediated ALSS differential expression of molecular players involved in JNK/Bsk mediated stress response pathway in Drosophila model of PD. In the PD brain Bsk and dFOXO expression was inhibited during both HP and TP. CU intervention differentially regulates Bsk-dFOXO axis-mediated stress response through the rescue of diminished dFOXO only during HP (a,b). However, with CU intervention expression of dFOXO downstream targets l(2)efl (Unaltered during HP and inhibited during TP upon PQ treatment) and 4e-bp (Upregulated upon PQ treatment in both HP and TP) remain unaltered (Compared to PD brain) during both the adult life stages (c,d). CU intervention mediated HP -specific rescue of upstream dFOXO, resulted in rescue of diminished GADD45 level (Compared to PD brain) durin g HP, whereas during TP expression of the same is further upregulated (Compared to PD brain) with CU intervention (e). Further, CU -intervention differentially modulates Bsk downstream HSP68 by inhibiting the upregulation (Compared to PD brain) during HP bu t not during TP (f). Similarly, CU intervention also differentially modulates Bsk downstream Puc (Inhibited during HP and unaltered during TP upon PQ treatment) through the rescue of diminished Puc level (Compared to PD brain) during HP (g). On the other h and, expression of dFOXO downstream antioxidant genes SOD1 and SOD2 (Unaltered during HP and inhibited during TP upon PQ treatment) were unaltered (Compared to PD brain) with CU intervention during both the adult life stages, whereas expression of CAT (Upregulated upon PQ treatment during both HP and TP) was inhibited with CU intervention during both the adult life stages (h,i,j). Overall insights suggest that CU -mediated preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 19 ALSS neuroprotection underlies differential modulation of dFOXO, dFOXO driven GADD45, HSP68 and Puc of the Bsk signalling pathway. Significance was drawn by analysing the data of a minimum of three replicates with one-way ANOVA followed by Tukey post hoc test. [ *p<0.05; **p<0.01; ***p<0.001; NS : Not significant - compared to PQ treated group ], [*C p<0.05; **C p<0.01; ***C p<0.001 - compared to Control (CTR) group]. 3.1.3. l(2)efl expression in the PD brain was diminished only during TP and CU fails to alter the same l(2)efl is regulated by dFOXO signalling and codes for an sHSP (small heat shock protein) of the α-crystallin family in Drosophila and is actively involved in promoting neuroprotection and longevity [28,45]. In the present study, l(2)efl expression in the PD brain was unaltered during HP but inhibited by 46% (P<0.05) during TP ( Fig 1c). CU intervention did not alter the diminished expression of l(2)efl during TP. However, CU per se feeding upregulated l(2)efl expression level by 2 fold (Compared to control brain) (P<0.001) during HP but not during TP ( Fig 1c). The observation suggests that CU can modulate l(2)efl expression only during the physiologic condition of HP. Further l(2)efl expression did not alter with age (Fig S1c). 3.1.4. CU has no influence on upregulated 4e-bp expression in the PD brain dFOXO signalling regulated 4e-bp in Drosophila codes for a protein that inhibits translation under stress conditions, thereby indirectly preventing protein aggregation and energy consumption [28, 46]. In the present study, 4e-bp expression in the PD brain was upregulated by 2 fold (P<0.001) during HP and by 4 fold (P<0.001) during TP ( Fig 1d). CU intervention did not alter the upregulated expression of 4e-bp during both adult life stages. However, CU per se feeding upregulated 4e-bp expression level by 44% (Compared to the control brain) (P<0.01) during HP but not during TP ( Fig 1 d). The observation suggests that CU can modulate 4e-bp expression only during the physiologic preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 20 condition of HP. Further, 4e-bp expression was also upregulated with aging [58% (P<0.001) higher in TP compared to HP] (Fig S1d). 3.1.5. CU rescues diminished GADD45 level in the PD brain only during HP dFOXO/FOXO signalling regulated GADD45 modulates DNA damage repair and can promote neuroprotection and apoptosis in Drosophila and in-vitro neuronal cell s respectively [47-51]. In the present study, GADD45 expression in the PD brain was inhibited by 20% (P<0.01) during HP, but upregulated by 72% (P<0.01) during TP ( Fig 1e). However, CU intervention rescued the diminished GADD45 level significantly (P<0.001) during HP, and it further enhanced (P<0.05) the upregulated GADD45 during TP (Fig 1e). The observation suggests that CU-mediated ALSS neuroprotection underlies the rescue of diminished GADD45 level during HP . Further investigation revealed that GADD45 expression is upregulated with aging [3 fold (P< 0.001) higher in TP compared to HP] (Fig S1e). 3.1.6. HSP68 was upregulated in the PD brain but CU rescues only during HP Bsk/JNK signalling regulates HSP and HSP68 may not be different. Its molecular chaperone-mediated response promotes longevity and stress resistance [27,29,52]. In the present study, HSP68 expression in the PD brain was upregulated by 2.5 fold (P<0.001) during HP and by 4 fold (P<0.01) during TP ( Fig 1 f). However, CU intervention significantly inhibited (P<0.001) the upregulated HSP68 level during HP but not during TP. The observation suggests that CU intervention can inhibit proteinopathic stress and molecular dysregulations associated with NDD. Therefore, CU-mediated ALSS neuroprotection also involves the rescue of altered HSP68 level. The investigation also revealed HSP68 expression is upregulated with aging [2 fold (P<0.05) higher in TP compared to HP] (Fig S1f). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 21 3.1.7. Puc expression diminished in the PD brain only during HP and CU rescues Hyper-activation of Bsk signalling is prevented by the expression of Bsk-phosphatase Puc through a negative feedback loop [29,53-55]. In the present study, Puc expression in the PD brain was inhibited by 38% (P<0.05) during HP, but expression of the same remained unaltered during TP. However, CU intervention rescued the diminished Puc level significantly (P<0.01) during HP ( Fig 1 g). Further, CU per se feeding also upregulated Puc expression by 83% (Compared to the control brain) (P<0.001) during HP but not during TP (Fig 1g). The observation suggests that Puc expression modulation in PD and physiologic conditions is a feature of CU, that is restricted only to HP. Therefore, CU-mediated ALSS neuroprotection underlies the modulation of Puc. Investigation revealed that Puc expression is downregulated with aging [51% (P<0.001) lower in TP compared to HP] (Fig S1g). 3.1.8. SOD1 and SOD2 diminished only during TP and CU fails to rescue dFOXO/FOXO signalling regulated SOD1 and SOD2 neutralizes superoxide in cytosol and mitochondria respectively and promotes neuroprotection and longevity [56-61]. In the present study, SOD1 and SOD2 expression in the PD brain was unaltered during HP but were inhibited by 52% (P<0.001) and 49% (P<0.001) respectively during TP ( Fig 1 h,i). CU intervention did not alter the diminished SOD1 and SOD2 levels during TP. The observation suggests that CU intervention has no role in modulating SOD1 and SOD2 in PD. It was observed that, while SOD1 expression is unaltered with aging and SOD2 expression is downregulated in the TP brain by 40% (P<0.01) compared to HP ( Fig S1 h,i). 3.1.9. CU inhibits upregulated CAT in the PD brain during both HP and TP preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 22 dFOXO/FOXO signalling regulated CAT expression is induced under stress as defense mechanism against peroxide and it also promotes longevity [56,57,59-63]. In the current study, CAT expression in the PD brain was upregulated by 35% (P<0.001) during HP and by nearly 2 fold (P<0.001) during TP ( Fig 1j). However, CU intervention significantly inhibited (P<0.001) the upregulated CAT level during both the adult life stages. Further, feeding of CU per se inhibited CAT expression by 56% (P<0.001) during HP and by 30% (P<0.05) during TP (Compared to the control brain) ( Fig 1j). The observation suggests that CAT expression modulation in PD and physiologic conditions is a feature of CU and is not restricted to the adult life stages. The ability of CU intervention to inhibit CAT upregulation in the PD brain signifies inhibition of peroxide stress, but TP flies are not rescued with CU intervention. Therefore, modulation of CAT may be necessary, but is not the critical to ALSS neuroprotection. It was observed that CAT expression in aging brain is unaltered (Fig S1j). 3.2. CU-mediated ALSS modulation of IIS-dTOR Signalling pathway 3.2.1. ILP2, ILP5 expression diminished in the PD brain while ILP3 was unaltered, but CU inhibits all the three ILP during HP and TP Drosophila ILP s are analogous to mammalian insulin/insulin-like signalling pathway (IIS), mediating anabolic processes and ablation of IIS components extends lifespan and enhance stress resistance [31,64-67]. In the present study, ILP2 expression in the PD brain was inhibited by 19% (P<0.001) during HP and by 17% (P<0.01) during TP. Diminished expression in the PD brain was also observed for the ILP5 which was inhibited by 31% (P<0.001) during HP and by 29% (P<0.01) during TP. ILP3 expression however was not altered in the PD brain of both life phases ( Fig 2 a ,b,c). CU preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 23 intervention further diminished the expression of ILP2 and ILP5 during HP (P<0.05 and P<0.01 respectively) and TP (P<0.01 and P<0.05 respectively). CU intervention also inhibited ILP3 expression during HP (P<0.01) and TP (P<0.001). The observation suggests that although only ILP2 and ILP5 are suppressed in the PD brain, CU intervention may promote suppression of IIS through the inhibition of all three ILPs. Inhibition of ILPs is therapeutic to organisms during stress, but transition phase flies are not rescued with CU intervention. Therefore, ILPs modulation in the PD brain may be a common mode of action of CU intervention and is not critically involved in ALSS neuroprotection. It is revealed that ILP2 expression was enhanced by 30% (P<0.001), ILP3 expression was diminished by 43% (P<0.01) and ILP5 expression was not altered with aging (Compared to HP) (Fig S2 a,b,c). Figure 2: CU-mediated ALSS differential expression of molecular players involved in IIS-dTOR mediated anabolic pathway in Drosophila model of PD. In the PD brain ILP2 and ILP5 expression was inhibited, whereas ILP3 expression remain unaltered during HP and TP. CU intervention inhibits IIS signaling by preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 24 further inhibiting ILP2, ILP5 and inhibiting ILP3 during both adult life stages (a,b,c). Similarly, CU intervention inhibits IIS signaling by inhibiting Chico (Upregulated during HP and TP upon PQ treatment) (d). Also, CU intervention inhibit s IIS downstream dTOR (Upregulated during HP and unaltered during TP upon PQ treatment) during both the adult life stages (e). But PD condition and CU intervention did not alter dTOR downstream RS6K1 expression (f). Overall insights suggest that CU intervention inhibits IIS- dTOR signaling during both the adult life stages. Though inhibition of IIS-dTOR inhibition is protective, TP flies are not rescued with CU intervention. Therefore, modulation of IIS-dTOR signaling is not critically involved in CU-mediated ALSS neuroprotection. Significance was drawn by analysing the data of a minimum of three replicates with one-way ANOVA followed by Tukey post hoc test. [*p<0.05; **p<0.01; ***p<0.001; NS: Not significant - compared to PQ treated group]. 3.2.2. CU rescues upregulated Chico in the PD brain during HP and TP Drosophila Chico codes for mammalian analogous the insulin receptor substrate for the propagation of IIS and inhibition of Chico also promotes life span extension cum stress resistance [31,64,68,69]. In the present study, Chico expression in the PD brain was upregulated by 2 fold (P<0.001) during HP and by 56% (P<0.05) during TP ( Fig 2d). However, CU intervention significantly inhibited the upregulation during both the HP (P<0.001) and TP (P<0.01) ( Fig 2d). The observation suggests that in both the adult life stages enhanced IIS signalling in PD condition is inhibited by CU intervention through inhibition of upregulated Chico. Inhibition of Chico is therapeutic to organisms under stress conditions, but TP flies are not rescued with CU intervention. Therefore, Chico modulation by CU intervention is not critically associated with ALSS neuroprotection. It was observed that natural aging enhances brain Chico level [2 fold (P<0.01) higher in TP compared to HP] (Fig S2d). 3.2.3. PD upregulated dTOR during HP and CU rescues, whereas in the TP PD brain dTOR expression was unaltered but CU inhibits the same IIS signalling-mediated activation of dTOR signalling ensures anabolic functions and reduced dTOR signalling promotes life span extension cum stress resistance [31,32,64,70]. In the present study, dTOR expression in the PD brain was enhanced by 88% (P<0.001) during HP, whereas no alteration was observed during TP ( Fig 2e). CU preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 25 intervention significantly inhibited the upregulation during HP (P<0.001) and inhibited dTOR level (P<0.05) during TP ( Fig 2e). The observation suggests that CU intervention inhibits dTOR signalling in PD brain. Inhibition of dTOR is therapeutic under stress conditions, but TP flies are not rescued with CU intervention. Therefore, dTOR modulation in the PD brain by CU is not critically associated with ALSS neuroprotection. It was observed that dTOR level in the brain is diminished with aging [21% (P<0.05) lesser in TP compared to HP] (Fig S2e). 3.2.4. RS6K1 expression was not altered in the PD brain and with CU intervention dTOR signalling also involves the activation of kinase coded by RS6K1 and similarly, its inhibition promotes life span extension cum stress resistance [31,32,64,70]. In the present study, RS6K1 expression was not altered in the PD condition or with CU intervention (Fig 2 f). The observation suggests that CU-mediated ALSS neuroprotection do es not involve RS6K1 modulation. It was observed that RS6K1 expression is diminished with aging [64% (P<0.001) lesser in TP compared to HP (Fig S2f)]. 3.3. CU-mediated ALSS modulation of components involved in mitochondrial dynamics 3.3.1. CU fails to rescue diminished Ewg expression in the PD brain during HP and TP IIS-dTOR/mTOR-mediated anabolic processes initiate mito-biogenesis th rough transcription factor NRF1/Ewg [71,72]. In the present study, Ewg expression in the PD brain was inhibited by 38% (P<0.01) during HP and by 24% (P<0.05) during TP ( Fig 3a). CU intervention did not alter the diminished expression of Ewg during both the adult life stages ( Fig 3a). The observation suggests that CU intervention has no influence in modulating Ewg in the PD brain of both adult life stages. Therefore, CU-mediated ALSS preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 26 neuroprotection do not underlie Ewg modulation. The investigation also revealed that Ewg expression is inhibited with natural aging [25% (P<0.001) lesser in TP compared to HP] (Fig S3a). 3.3.2. TFAM expression was unaltered in the PD brain, but CU intervention inhibits during HP and TP Ewg signalling regulates TF AM mediating mtDNA replication for mito-biogenesis ( 71- 73). In the present study, TF AM expression in the PD brain was not altered during HP and TP (Fig 3 b). However, CU intervention significantly inhibited TF AM level during HP (P<0.001) and TP (P<0.01). Further, feeding of CU per se inhibited TF AM expression by 47% (P<0.001) during HP and by 62% (P<0.001) during TP ( Fig 3b). The observation suggests that TF AM modulation in PD and physiologic conditions is a feature of CU, which is common to both the adult life stages. TF AM inhibition may be protective, but TP flies are not rescued with CU intervention. Therefore, modulation of TF AM is not critically involved in CU-mediated ALSS neuroprotection. The investigation also revealed that TF AM expression is diminished with natural aging [30% (P<0.001) lesser in TP compared to HP] (Fig S3b). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 27 Figure 3 : CU-mediated ALSS differential expression of molecular players involved in mitochondrial dynamics in the Drosophila model of PD. In the PD brain mito-biogenesis mediating transcription factor Ewg expression was inhibited during both HP and TP and CU intervention fails to alter the diminished Ewg level (a). However, with CU intervention Ewg downstream TFAM level (Unaltered upon PQ treatment) was inhibited during both HP and TP (b). In the PD brain Mfn2 level was inhibited during both HP and TP. CU differentially regulates mitochondrial quality control through the rescue of diminished Mfn2 level during HP, but not during TP (c). Similarly, CU intervention differentially regulates mitochondrial respiratory capacity, through upregulating ATP SynD (Unaltered upon PQ treatment) level only during HP (d). Significance was drawn by analysing the data of a minimum of three replicates with one-way ANOVA followed by Tukey post hoc test. [ *p<0.05; **p<0.01; ***p <0.001; NS: Not significant - compared to PQ treated group], [*C p<0.05; **C p<0.01; ***C p<0.001 - compared to Control (CTR) group]. 3.3.3. Mfn2 level was diminished in the PD brain and CU intervention rescues only during HP dFOXO/FOXO signalling regulates Mfn2 to mediate mitochondrial quality control, complementation and it actively promotes neuroprotection [33,34,74-76]. In the present study, Mfn2 expression in the PD brain was inhibited by 39% (P<0.05) during HP and by 24% (P<0.05) during TP ( Fig 3c). However, CU intervention rescued diminished Mfn2 level significantly (P<0.01) only during HP but not during TP. Feeding of CU per se also upregulated Mfn2 expression by nearly 2 folds (P<0.001) only during HP ( Fig 3c). The observation suggests that Mfn2 expression modulation in PD and physiologic conditions preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 28 is a feature of CU that is restricted only to HP. Therefore, CU mediated ALSS neuroprotection underlies modulation of Mfn2. It was found that Mfn2 expression is enhanced with aging [20% (P<0.01) higher in TP compared to HP] (Fig S3c). 3.3.4. ATP SynD expression was unaltered in the PD brain but CU intervention upregulates only in HP ATP SynD codes for the ATP synthase subunit associated with the Complex V (Cl-V) of the mitochondrial respiratory chain [77]. In the present study, ATP SynD expression in the PD brain was not altered during HP and TP. CU intervention upregulated ATP SynD expression (P<0.05) only during HP only but not during TP ( Fig 3d). Also, feeding of CU per se upregulated ATP SynD expression by nearly 2 folds (P<0.001) during HP and by 43% (P<0.001) during TP ( Fig 3 d). The observation suggests that CU-mediated modulation of ATP SynD under the PD condition is restricted only to HP. Further, the PD condition might not specifically affect Cl-V of the respiratory chain, but CU-mediated differential modulation of ATP SynD may contribute to better respiratory health during HP. It was observed that ATP SynD downregulates with natural aging [70% (P<0.001) lesser in TP compared to HP] (Fig S3d). 3.4. CU-mediated ALSS modulation of phase II ADS 3.4.1. CncC was diminished in the PD brain and CU intervention rescues only during HP CncC (Mammalian Nrf2) actively participates in the regulation of phase II ADS and is a potential target of neuroprotection in PD [78-80]. In the present study, CncC expression in the PD brain was inhibited by 55% (P<0.001) during HP and by 51% (P<0.01) during TP (Fig 4 a). However, CU intervention rescued (P<0.01) diminished CncC level only preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 29 during HP but not during TP (Fig 4a). The observation suggests that CU-mediated ALSS neuroprotection underlies HP-specific rescue of CncC. Further, the investigation revealed that CncC expression is upregulated with natural aging [2 fold (P<0.001) higher in TP compared to HP] (Fig S4a). 3.4.2. CU rescues diminished GCLC level in PD brain only during HP CncC-mediated regulation of phase II ADS and neuroprotection also involves the production and utilization of reduced GSH which is mediated by GCLC [78,81,82]. In the present study, GCLC expression in the PD brain was inhibited by 42% (P<0.001) during HP, but upregulated by 63% (P<0.001) during TP ( Fig 4b ). However, CU intervention rescued (P<0.05) the diminished GCLC level during HP, but failed to alter GCLC upregulation during TP ( Fig 4 b). The observation suggests that CU-mediated ALSS neuroprotection underlies the rescue of diminished GCLC level during HP. Further investigation revealed that GCLC expression is diminished with aging [50% (P<0.001) lesser in TP compared to HP] (Fig S4b). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 30 Figure 4: CU-mediated ALSS differential expression of molecular players involved in phase II ADS in the Drosophila model of PD. In the PD brain CncC expression was inhibited during both HP and TP. CU intervention differentially regulates phase II ADS through the rescue of diminished CncC only during HP (a). CU intervention mediated HP -specific rescue of upstream CncC resulted in the rescue of diminished GCLC level (Compared to PD brain) during HP, whereas during TP expression of upregulated GCLC was not altered with CU intervention (b). Further, CU -intervention differentially modulates CncC downstream GCLM by inhibiting the upregulation (Compared to PD brain) during HP but not during TP (c) CU intervention did not alter CncC downstream GSTD1 (Upregulated upon PQ treatment during HP and TP) expression level (d). Further, CU -intervention differentially modulates CncC downstream Prx 2540-1,2 by inhibiting the upregulation (Compared to PD brain) during HP but not during TP (e). CU intervention mediated HP -specific rescue of upstream CncC resulted in the rescue of diminished Jafrac1 level (Compared to PD brain) d uring HP, whereas during TP expression of the same was further upregulated with CU intervention (f). However, Jafrac2 was not altered in the PD brain or with CU intervention (g). The diminished level of CncC downstream Prx5 was rescued (Compared to PD brai n) with CU intervention during HP and TP (h). However, the diminished level of Prx3 was rescued (Compared to PD brain) with CU intervention only during HP, like upstream CncC (i). Overall insights suggest that CU - mediated ALSS neuroprotection underlies dif ferential modulation of phase II ADS mediator CncC, which

in synchronous differential modulation of Prx3. Rescue of diminished CncC during HP also results in synchronous rescue of diminished GCLC and Jafrac1. CU -mediated ALSS neuroprotection also underlies differential modulation of GCLM and Prx 2540-1,2 which is not synchronous to upstream CncC. Significance was drawn by analysing the data of a minimum of three replicates with one -way ANOVA followed by Tukey post hoc test. [*p<0.05; **p<0.01; ***p< 0.001; NS: Not significant - compared to PQ treated group], [*C p<0.05; **C p<0.01; ***C p<0.001 - compared to Control (CTR) group]. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 31 3.4.3. GCLM upregulated in PD brain but CU rescues only during HP CncC-mediated regulation of GCLM ensures the stability of the GSH synthesis machinery and its gene response is necessary for the prevention of neurodegeneration [82-84]. In the present study, GCLM expression in the PD brain was upregulated by 70% (P<0.001) during HP and by 3 fold (P<0.001) during TP ( Fig 4 c). However, CU intervention significantly inhibited (P<0.001) the upregulated GCLM level during HP but not during TP ( Fig 4c). The observation suggests that CU intervention can inhibit some form of stress that is specifically countered by phase II ADS, normalizing the associated molecular dysregulation. Therefore, CU-mediated ALSS neuroprotection involves modulation of GCLM. Further, investigation revealed that GCLM expression is diminished with aging [25 % (P<0.01) lesser in TP compared to HP] (Fig S4c). 3.4.4. CU does not alter the upregulated GSTD1 expression in PD brain CncC and Bsk signalling regulated GSTD1 mediates toxin neutralization through reduced and it is often upregulated in the presence of toxins cum xenobiotics [29,62, 80,85-88]. In the present study, GSTD1 expression in PD brain was upregulated by 36% (P<0.01) during HP and by 80% (P<0.01) during TP ( Fig 4d). CU intervention did not alter the upregulated expression of GSTD1 during both the adult life stages. The observation suggests that CU do not modulate GSTD1 expression in the PD brain. Therefore, CU- mediated ALSS neuroprotection do es not underlie GSTD1 modulation. Further, GSTD1 expression was also enhanced with aging [80% (P<0.001) higher in TP compared to HP] (Fig S4d). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 32 3.4.5. Prx 2540-1,2 was upregulated in the PD brain but CU rescues only during HP Prx 2540-1,2 is a copy variant of the Prx 2540 and may be regulated by CncC signalling and it also mediates pro-inflammatory pathways [8 9-91]. In the present study, Prx 2540- 1,2 expression in PD was upregulated by 70% (P<0.01) during HP and by 4 fold (P<0.001) during TP (Fig 4e). However, CU intervention significantly inhibited (P<0.01) the upregulated Prx 2540-1, 2 level only during HP but not during TP ( Fig 4 e). The observation suggests that CU intervention can inhibit some form of stress that is specifically countered by phase II ADS and/or inhibit pro-inflammatory signature by inhibiting associated molecular dysregulation. Therefore, CU-media ted ALSS neuroprotection involves modulation of Prx 2540-1,2 expression. Further, investigation also revealed that Prx 2540-1, 2 expression is upregulated with aging [2 fold (P<0.01) higher in TP compared to HP] (Fig S4e). 3.4.6. CU rescues diminished Jafrac1 level in the PD brain only during HP Jafrac1 is regulated under CncC and Bsk-dFOXO signalling and is actively involved in neuroprotection and stress resistance [90,92,93]. In the present study, Jafrac1 expression in PD brain was inhibited by 24% (P<0.05) during HP, but upregulated by 70% (P<0.05) during TP (Fig 4f). However, CU intervention rescued diminished Jafrac1 level significantly (P<0.001) during HP, but during TP the upregulation of Jafrac1 was further enhanced (P<0.001) (Fig 4f). The observation suggests that CU -mediated ALSS neuroprotection underlies the rescue of diminished Jafrac1 level during HP . Further, the investigation revealed that Jafrac1 expression is diminished with aging [56% (P<0.001) lesser in TP compared to HP] (Fig S4f). 3.4.7. Jafrac2 expression was not altered in the PD brain and with CU intervention preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 33 Jafrac2 is regulated under CncC signalling and promotes longevity, stress resistance and also promotes apoptosis depending on its signalling intensity [90,94]. In the present study, Jafrac2 expression was not altered in PD brain and with CU intervention, during both the adult life stages ( Fig 4g). No alteration in expression was also obser ved upon CU per se feeding during both the adult life stages ( Fig 4g). The observation suggests that CU-mediated ALSS neuroprotection do es not underlie Jafrac2 modulation. Jafrac2 expression is upregulated with aging [68% (P<0.001) higher in TP compared to HP] (Fig S4g). 3.4.8. CU rescues diminished Prx5 expression in the PD brain during HP and TP CncC-regulated Prx5 codes for mitochondria and cytosol-specific peroxiredoxin that extensively promotes longevity, apoptosis prevention and anti-inflammatory response [90, 95-97]. In the present study, Prx5 expression in the PD brain was inhibited by 26% (P<0.05) during HP and by 42% (P<0.001) during TP (Fig 4h). However, CU intervention rescued diminished Prx5 level significantly (P<0.001) during both the adult life stages (Fig 4h). But TP flies are not rescued with CU intervention. Therefore, Prx5 modulation by CU in the PD brain may be important but is not cr itical to ALSS neuroprotection. Further investigation revealed that Prx5 expression is upregulated with aging [25% (P<0.01) higher in TP compared to HP] (Fig S4h). 3.4.9. Prx3 level was diminished in the PD brain and CU intervention rescues only during HP CncC and dFOXO/FOXO regulated Prx3 codes for only mitochondria-specific peroxiredoxin that promote longevity, stress resistance cum neuroprotection [49,90 ,96]. In the present study, Prx3 expression in the PD brain was inhibited by 50% (P<0.001) during HP and by 30% (P<0.001) during TP ( Fig 4i). However, CU intervention rescued preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 34 (P<0.01) diminished Prx3 level only during HP but not during TP ( Fig 4i). The observation suggests that CU -mediated ALSS neurop rotection underlies the modulation of Prx3. Further investigation also revealed that Prx3 expression is upregulated with aging [30% (P<0.001) higher in TP compared to HP] (Fig S4i). 3.5.CU-mediated ALSS modulation of components involved in metal homeostasis 3.5.1. CU partially rescues the upregulated MtnA in the PD brain MtnA is responsible for zinc/copper homeostasis that promotes anti-inflammatory, anti- oxidant responses against stress [98,99-102]. In the present study, MtnA expression in the PD brain was upregulated by 2 fold (P<0.001) during HP and by nearly 3 fold (P<0.001) during TP (Fig 5a). However, CU intervention partially inhibited the MtnA upregulation (P<0.001) during both the adult life stages. Also, CU per se feeding inhibited MtnA expression by 60% (P<0.001) during HP and by 40% (P<0.001) during TP ( Fig 5a). The observation suggests that MtnA expression modulation in the PD and physiologic conditions is a feature of CU, which is common to both the adult the life stages. The ability of CU to partially inhibit MtnA upregulation in the PD brain suggests its ability to suppress oxidative stress, inflammation and associated molecular dysregulation. But TP flies are not rescued with CU intervention, suggesting modulation of MtnA does not underlie CU-mediated ALSS neuroprotection. Further, MtnA expression did not alter with aging (Fig S5a). 3.5.2. CU partially rescues the upregulated Fer1HCH in the PD brain Fer1HCH codes for an iron-chelating protein and is necessary for neuronal maintenance cum CNS development in Drosophila [103-106]. In the present study, Fer1HCH expression in PD brain was upregulated by 3 fold (P<0.001) during HP and by 2.6 fold (P<0.001) during TP ( Fig 5b). However, CU intervention partially inhibited Fer1HCH preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 35 upregulation during HP (P<0.05) and TP (P<0.001) ( Fig 5b). The observation suggests the ability of CU to suppress iron dysregulation, and oxidative stress and inhibit associated molecular signature. But TP flies are not rescued with CU intervention, suggesting modulation of Fer1HCH does not underlie ALSS neuroprotection. Further, Fer1HCH expression was enhanced with aging [2.6 folds (P<0.001) higher in TP compared to HP] (Fig S5b). Figure 5: CU-mediated ALSS differential expression of molecular players involved in metal homeostasis in the Drosophila model of PD. In the PD brain MtnA and Fer1HCH expression was upregulated during both the HP and TP. CU intervention partially inhibits the PD-associated upregulation of MtnA and Fer1HCH during both the adult life stages (a,b). CU intervention in PD brain did not alter IRP-1A and IRP- 1B (Unaltered during HP and upregulated during TP upon PQ treatment) during HP but during TP it inhibits IRP dysregulation by significantly inhibiting IRP-1A (Compared to PD brain) and partially inhibiting IRP-1B (Compared to PD brain) (c,d). Overall insights suggest that owing to anti-inflammatory, antioxidant and metal-chelating properties CU in the PD brain can partially suppress possible metal (Zinc, copper and iron) imbalance and partially inhibits the dysregulation of MtnA and Fer1HCH. However, CU cannot fully suppress iron intake during TP as it can only inhibit IRP-1A upregulation completely, whereas upregulated IRP-1B is inhibited partially in PD brain The. limitation of CU to prevent iron intake in the PD brain during TP highlights its limitation to promote neuroprotection during later adult life stages. Significance was drawn by analysing the data of a minimum of three replicates with one-way ANOVA preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 36 followed by Tukey post hoc test. [ *p<0.05; **p<0.01; ***p<0.001; NS : Not significant - compared to PQ treated group], [*C p<0.05; **C p<0.01; ***C p<0.001 - compared to Control (CTR) group]. 3.5.3. IRP-1A expression in the PD brain was upregulated only during TP and CU inhibits the same IRP-1A like its mammalian counterpart IRP1 initiates iron uptake in cells, whereas high IRP1 signalling activity is already established as a potent cause of proteinopathic NDD like AD and PD [107-110]. In the present study, IRP-1A expression in the PD brain is not altered during HP but upregulated by 45% (P<0.001) during TP ( Fig 5c). CU significantly inhibited (P<0.001) the IRP-1A upregulation ( Fig 5c). The observation suggests that during TP, CU intervention inhibits iron uptake by modulating IRP-1A, but it may not contribute to neuroprotection. Further, IRP-1A expression was upregulated with aging [Nearly 10 fold (P<0.001) higher in TP compared to HP] (Fig S5c). 3.5.4. IRP-1B expression in PD brain was upregulated only during TP and CU partially rescues IRP-1B similar to IRP-1A is responsible for iron intake and may also promote the progression of NDD like AD and PD [107-110]. In the present study, IRP-1B expression in the PD brain was not altered during HP but upregulated by 77% (P<0.001) during TP (Fig 5d). CU intervention partially inhibits (P<0.001) the IRP-1B upregulation (Fig 5D). The observation suggests that during TP, CU intervention inhibits iron uptake by modulating IRP-1B, but it may not contribute to neuroprotection. Further, IRP-1B expression was upregulated with aging [70% (P<0.001) higher in TP compared to HP] (Fig S5d). preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 37 4. Discussion To understand the molecular basis of ALSS DAergic neuroprotective efficacy of CU we made a systematic hard effort by looking into the brain-specific modulation of molecular targets and pathways that are implicated in human DAergic neurodegeneration/neuroprotection viz., a) dFOXO, GADD45, PUC and proteinopathic stress under Bsk-signalling mediated adaptive stress response pathway; b) IIS-dTOR mediated anabolic signaling, mitochondrial quality control under JNK-FOXO and IIS- TOR antagonism-mediated mitochondrial dynamics; c) CncC, GCLC, Prx 2540, Jafrac1 and Prx3 of the Phase II antioxidant defense system; d) MtnA, Fer1HCH, IRP-1A and IRP-1B mediated brain iron metabolism. Our comprehensive work illustrates that: 4.1 CU-mediated ALSS neuroprotection underlies HP-specific modulation of dFOXO, GADD45, PUC and proteinopathic stress under Bsk-signalling mediated adaptive stress response pathway Bsk-signalling contributes to neuronal survival, and longevity by activating antioxidant response, heat shock response, autophagy and insulin signalling antagonism, when activated in moderation [25-29], but its hyperactivation/overexpression also promotes apoptosis [25,27,111 ]. Further, the Bsk-dFOXO signalling axis is necessary for the promotion of cell survival through adaptation to stress by activating heat shock, endogenous antioxidants, growth arrest, DNA damage repair and autophagic response [28]. In the present study, Bsk expression in the PD brain was repressed during both the HP and TP (Fig 1a). It was reported that ubiquitous repression of JNK signalling in a Bsk mutant (EMS-induced) Drosophila model enhances susceptibility to PQ-induced stress [29]. On the other hand, it was demonstrated in Drosophila that neuronal enhancement of Bsk signalling (Heterozygous loss of Puc and Bsk overexpression) promotes resistance to preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 38 PQ-induced stress, enhanced longevity, and delayed neuronal senescence [26, 29] . Concurring w ith the previous reports, it can be postulated in the present study that depleted Bsk expression is associated with PQ-mediated DAergic neuronal dysfunction and onset of PD during both the adult life stages. However, failure of CU to correct diminished Bsk level suggests, CU mediated HP-specific neuroprotection is not through Bsk modulation. Yet Bsk active form is the phosphorylated protein, hence further insight is needed to comment on the role of this entity in ALSS neuroprotection. Downstream dFOXO expression was also diminished in PD brain of both life stages similar to upstream Bsk. However, it was rescued with CU intervention only during HP, but not during TP ( Fig 1b). Tas et al, [43], reported that inhibition of dFOXO signalling through RNAi or deletion spanning in the dFOXO gene (Heterozygous or homozygous condition) promotes the loss of DAergic neurons and selective loss of PAM DA neuronal cluster. Therefore, onset of PD in this scenario may be associated with downregulation of dFOXO. PINK1 null flies have shown significant DAergic degeneration, and mitochondrial dysfunction in the brain and thorax which was rescued with dFOXO and downstream gene ( SOD2, 4e-bp) overexpression [44]. Further, CU-analogue tetra-hydro curcumin (THC) mediated life span extension and stress resistance requires the presence of functional dFOXO [112]. It is hypothesized and reported that phenotypes like life span extension and DAergic neuroprotection may go hand in organisms [12, 113]. Therefore, it can be postulated from the current study that CU-mediated HP-specific neuroprotection involves dFOXO modulation which further down the line might modulate its target gene(s) of neuroprotection. CU per se in the current study was demonstrated to enhance Bsk and dFOXO level (Compared to the control brain) only during HP ( Fig 1 a ,b). This signifies enhanced dFOXO signalling with CU under the physiologic condition. CU is reported as an early- preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 39 acting inducer of longevity, as it enhances fly life span when fed at early stages or during HP of life through dietary means [114 , 115]. Therefore, from the current study, it can be put forward that HP-specific CU-mediated modulation of Bsk-dFOXO during physiologic condition may contribute to longevity or better physiology. However, further insight is needed. Limitation of CU to modulate dFOXO during both PD and physiologic condition, Bsk during physiologic condition in TP may be due to diminished level of both the Bsk and dFOXO in a healthy aging brain ( Fig S1 a ,b), suggesting hindrance of their regulatory system with natural aging. It corroborates with the reports suggesting that with senescence dFOXO signalling activity in the muscle and neuron declines [116]. dFOXO-mediated GADD45 regulation can initiate the protective DNA damage repair response by arresting cell growth and in some cases GADD45 can also induce the pro - apoptotic signal in neuronal cells. The result demonstrated that in the PD brain of HP, GADD45 expression is inhibited significantly ( Fig 1e ). Maitra et al, [117] (Supplementary data) reported that targeted RNAi-mediated knockdown of GADD45 in DAergic neurons increases lethality of HP Drosophila to PQ exposure. Hence, it can be postulated that the downregulation of GADD45 might underlie the onset of PD during HP. The current study reports that during HP diminished GADD45 level is rescued with CU intervention (Fig 1e) similar to upstream dFOXO (Fig 1b). Conditional GADD45 overexpression (4 folds) in the fly nervous system was demonstrated to enhance stress resistance against PQ [48,50]. Therefore, CU mediated GADD45 modulation driven by rescue of upstream dFOXO may contribute to life stage specific neuroprotection. However, contrasting to HP, TP PD brain demonstrated significantly increased GADD45 expression, which was further elevated with CU intervention ( Fig 1e ). Considering the protective nature of GADD45, this upregulation may be a TP-specific stress compensatory response which was further boosted with CU preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 40 intervention and is not correlated to upstream dFOXO ( Fig 1b ). But TP flies are not rescued from parkinsonian symptoms with CU intervention, suggesting the upregulation is not associated with neuroprotection. Interestingly it was reported that conditional GADD45 overexpression (4 folds) in fly nervous system, more than 48 hrs increases mortality of flies under acute PQ (20mM) induced stress [48]. Further, 4.2-7.2 fold and 2.5 -3.2 fold over expression of mammalian GADD45γ and GADD45β under nutrient deficiency was associated with neuronal apoptosis in-vitro [51]. Therefore, it is possible GADD45 overexpression promotes neuroprotection in a dose dependent manner under stress condition. As observed with aging GADD45 expression was elevated in fly brain (Fig S1e) that may superimpose on the upregulation of the same in TP PD brain. This may lead to negative consequence during later stages of life. Failure of CU to rescue the altered level of GADD45 in TP PD brain highlights its limitation as a therapeutic agent against late onset NDD like PD. HP specific modulation of Bsk-signalling thorough dFOXO-GADD45 won’t be enough to promote neuroprotection if Bsk signalling is not balanced. The intensity of Bsk signalling is carefully modulated by a negative feedback loop. Bsk signalling-mediated regulation of Puc under the negative feedback loop promotes dephosphorylation of p -Bsk, preventing Bsk signalling overactivation and apoptosis [29,53-55]. In the current study, the result demonstrated that during HP, Puc expression was diminished in the PD brain (Fig 1g ). It was reported that downregulation of DUSP6 and DUSP26 (A mammalian orthologue of Drosophila Puc), downregulation was associated with onset and progression of PD, reduced tyrosine hydroxylase, poor ATP generation, ROS production and impaired mitochondrial mobility (Rate limiting enzyme for dopamine synthesis) level [118 , 119 ]. Hence, it can be postulated that during HP, the onset of PD, is associated with diminished Puc level. Further, in fly models several independent studies preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 41 reported that overexpression of Puc suppressed irradiation and Alzheimers Disease- induced Bsk hyperactivation, alleviating morphological anomaly in wing and degenerative eye phenotype [54,55]. Therefore, it can be concluded that rescue of diminished Puc levels with CU in HP PD brain underlies DAergic neuroprotection . It was observed that similar to upstream Bsk ( Fig 1a ) CU per se upregulated Puc expression (Compared to the control brain) during HP ( Fig 1g ). It is evident that HP- specific modulation of Puc by CU is not limited to the PD condition, but is also present under the physiologic condition. Hence, it cannot be ruled out that CU mediated early acting longevity may also include modulation of Puc of the Bsk-signalling pathway under physiologic condition. The unresponsiveness of Puc during TP suggests under PD and CU intervention suggests, Bsk self-regulatory axis was not utilized in any condition. Further, it was observed that aging brain had a significantly lower Puc level ( Fig S1g ). This insight suggests natural aging-associated hindrance of the Puc regulatory mechanism. During TP such natural aging-associated changes might prevent utilization of the Bsk -Puc axis under various conditions. The regulation of HSP coding genes in general has been reported under the control of Bsk signalling and HSP68 may be no different. HSP68-mediated large heat shock response is responsible for clearing toxic aggregates and promotes protein refolding in cells. The HSP coded by HSP68 and its constitutive isoform Hsc70 act as a chaperone and protects the cell by binding to misfolded proteins induced by oxidative stress, preventing toxic aggregation proteins [120 ]. In the current study, it was demonstrated that in PD brain HSP68 expression was enhanced during both the adult life stage ( Fig 1f ). This corroborates with report of Maitra et al, [117] and considering the protective nature of the molecular player [121] suggesting neurotoxicant induces HSP68 expression possibly as a preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 42 stress compensatory response. Upon CU intervention, the altered level of HSP68 is rescued only during HP but not during TP ( Fig 1f). The ability of CU to inhibit HSP68 upregulation in the PD brain suggests that CU intervention can ablate proteinopathic stress (protein misfolding and aggregation) only during HP. The current study also demonstrated that HSP68 expression upregulated with aging ( Fig S1f ), suggesting induction of proteinopathic stress with natural aging. The onset of PD in the TP brain may further enhance cumulative proteinopathic stress. The failure of CU to impart neuroprotection in TP PD brain is due to its limitation in inhibiting proteinopathic stress. Hence, HSP68 upregulation is also not normalized in TP PD brain. Looking into other downstream targets of Bsk signalling it was found that 4e-bp and CAT expression was upregulated in PD brain of both life stages ( Fig 1 d, j). Neurotoxicant mediated upregulation of ubiquitous 4e-bp and brain specific CAT in adult-young flies were previously reported [63,122]. Corroborating with these reports from the current study it can be postulated that 4e-bp and CAT upregulation in PD brain are stress compensatory response. However, CU-intervention did not alter 4e-bp level in PD brain, whereas it inhibited altered level of CAT in PD brain of both life phases ( Fig 1 d,j). CU mediated inhibition of altered CAT level is possibly due to CU’s innate antioxidant capability. This corroborates with observation of Phom [23], suggesting although OS is sequestered during both adult life stages, it is not enough to promote neuroprotection during TP. Overall insight suggest CU-mediated HP specific neuroprotection is not through 4e-bp and CAT. Small heat shock protein l(2)efl and cytosolic and mitochondrial superoxide dismutase i.e. SOD1, SOD2 similar differential regulatory pattern. In HP PD brain, l (2)efl, SOD1 and SOD2 are not altered in PD brain or with CU intervention (Fig 1 c, h,i). No alteration of SOD1 and SOD2 under neurotoxicant exposure in adult young fly brain was preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 43 previously also reported [63]. However, in TP PD brain l(2)efl, SOD1 and SOD2 levels were diminished in PD brain and CU-intervention fails to rescue the same. In light of the neuroprotective and longevity extending aspect of l(2)efl, SOD1 and SOD2 [29,44,61], it is evident that in TP PD brain diminished level of the molecular players suggests susceptibility of TP brain to stress. Failure of CU to correct the diminished level of the players in TP PD brain highlights limitation of CU in protecting neurons at later stages of life. 4. 2 CU-mediated ALSS neuroprotection in not through modulation of IIS-dTOR mediated anabolic signalling Insulin/Insulin-like signalling ( IIS) governs nutrient-dependent organismal growth, anabolic process, neural growth/genesis, autophagy inhibition and protein translation through activation of TOR signalling. Yet during stress or later stages of life it is more feasible curb this nergy consuming anabolic pathways in favor of energy producing pathways, thereby reducing ROS generation [30]. The current study demonstrated that ILP s (Drosophila ortholougue of insulin and isnsulin like growth factor) show variable patterns of expressional changes independent of each other. In the PD brain ILP2 and ILP5 were significantly downregulated ( Fig 2 a ,c), whereas ILP3 expression was not altered during both the adult life stages ( Fig 2b). The observation agrees with report of Karpac et al., [123 ] in adult young fly, which suggests such pattern of inhibition of ILP2,5 and unaltered level of ILP3 is due to adaptive activation of JNK signalling pathway under neurotoxicant stress resulting in suppression of growth-related anabolic signalling. The current study also demonstrates that ILP downstream insulin receptor substrate Chico level was upregulated in the PD brain of both adult life stages (Fig 2d). A high sugar diet is reported to promote Type 2 Diabetes Mellitus in adult young (15 days old) Drosophila, resulting in ubiquitous upregulation Chico and induction of motor preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 44 deficit [124 ]. Therefore, it is possible upregulated Chico in the PD brain may lead to some form of metabolic consequences contributing to onset and progression of PD. CU intervention inhibited all the ILPs level and corrected alter Chico level in PD brain of both life stages ( Fig 2 a-d ). Aging, metabolic response/disorder and neurodegeneration go hand in hand and components of IIS actively regulate the process [31]. Ubiquitous inhibition of ILPs and Chico expression through RNAi mediated knock down and gene knock out respectively promotes extended lifespan, and stress resistance in fly models [69,125 ]. However, despite the positive implications of IIS i.e., ILPs and Chico inhibition TP flies are not rescued with CU intervention. It is possible that inhibition mediated longevity extension and DAergic neuroprotection do not go hand in hand, therefore CU mediated HP-specific neuroprotection may not be through modulation of IIS. To further understand the role of the anabolic signalling in ALSS neuroprotection we investigated the differential modulation of dTOR (Fly orthologue of mTOR) and RS6K1. The current study demonstrates that dTOR expression in PD brain was significantly enhanced during HP, whereas expression of the same was not altered during TP ( Fig 2e). In the adult young (2-3 months old) mice model, it was demonstrated that PQ exposure

in the onset of PD with an enhanced mTOR translate level [126 ]. The current observation in the HP PD brain corroborates with the finding of Wills et al, [126] suggesting higher dTOR transcript level may in fact be associated with the onset of PD. However, during TP such upregulation in PD is not observed, but the possibility of higher dTOR kinase activity cannot be ruled out as Chico of the upstream IIS signalling is upregulated in the TP PD brain ( Fig 2d ), suggesting enhanced signalling of IIS. CU intervention during HP normalizes dTOR altered level, whereas inhibits the same during TP ( Fig 2e ). Dietary intervention of CU is shown to promote life span extension in Drosophila through dTOR inhibition [127]. Further, ubiquitous overexpression of a preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 45 dominant negative form of dTOR increases the Drosophila life span [70]. However, flies of TP are not rescued with CU intervention from PD symptoms, therefore it is possible DAergic neuroprotection by CU does not underlie modulation of dTOR similar to upstream IIS. It must be noted however that the active form of IIS-dTOR pathway is phosphorylated Chico, dTOR and RS6K1. Hence, further insight is needed to comment on the role of this pathway in CU-mediated ALSS neuroprotection. 4.3 CU-mediated ALSS neuroprotection in through rescue of mitochondrial quality control under JNK-FOXO and IIS-TOR antagonism-mediated mitochondrial dynamics Anabolic processes require higher energy consumption; hence mito-biogenesis must be increased. Insights suggest that mTOR/dTOR signalling of the IIS-mTOR/dTOR pathway can also promote gene expression and translation of transcription factor coding Ewg (NRF1 as mammalian orthologue), which down the line regulates the expression of TF AM. TF AM promotes the replication and transcription of mtDNA necessary for mito- biogenesis [71,72]. The current study demonstrated that in the PD brain of both adult life stages, Ewg expression is downregulated and CU intervention fails to rescue ( Fig 3a ). Neurotoxicant like MPTP and MPP + (Chemically very similar to PQ) is reported to inhibit NRF1 expression, and translate level in neuronal cell culture and in mice brain resulting in PD condition [128 , 1 29]. The current observation in Drosophila PD brain corroborates the insights from Wang et al, and Piao et al, [128 , 1 29] suggesting that diminished Ewg is associated to onset of PD. As Ewg also expresses electron transport chain (ETC) subunits [130 ], diminished Ewg may also give cues to deficiency in ATP preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 46 production. Failure of CU to rescue deficient Ewg level during both life stages suggest, CU-mediated ALSS neuroprotection is not through Ewg. On the other hand, to obtain further insight into the mito-biogenesis under IIS-dTOR signalling, expression analysis of TF AM was also performed. The current study demonstrated that during both the adult life stages TF AM expression is not altered in the PD brain, yet CU intervention inhibited TF AM ( Fig 3b ). CU mediated inhibition of TF AM is like inhibition of upstream anabolic IIS-dTOR (Fig 2 a -e). In a mice model of AD, CU intervention had contrasting action on TF AM expression. It was demonstrated that depending on the mode of toxicity with two different variants of ApoE –4, CU either upregulated TF AM or downregulated it to promote neuroprotection and uplift mitochondrial health [131 ]. Hence, it is highly likely that CU- mediated rescue of mitochondrial biology through TF AM modulation is highly adaptive to the nature of toxicity and degenerative condition. Further, when mitochondrial biogenesis-specific signalling is enhanced, it leads to neural degeneration in the fly model. RNAi-mediated TF AM inhibition is demonstrated to be neuroprotective in such cases of aberrant mito- biogenesis [73,132]. In the current study, enhanced it is evident that during time of stress CU intervention also suppresses mito-biogenesis along with upstream IIS-dTOR anabolic signalling. This might have a protective aspect as it may prevent propagation of faulty mitochondrial DNA, but TP flies are not rescued with CU intervention. Therefore, CU- mediated ALSS neuroprotection is not through modulation of TF AM. With the insight on IIS-dTOR anabolic signalling mediated mito-biogenesis in ALSS neuroprotection, We further investigated the mitochondrial quality control antagonistic to mito-biogenesis and is controlled by Bsk-dFOXO pathway [33,34,74-76]. Like upstream dFOXO ( Fig 1b ), diminished level of Mfn2 in PD brain was rescued with CU- intervention only during HP but not during TP ( Fig 3c). In young fly model and mice preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 47 model neurotoxicant exposure resulted in diminished Mfn2 level (Transcript and translate) resulting in oxidative stress, motor deficit, mitochondrial fragmentation in thoraces and brain respectively [74,75]. PD patients also show a trend toward downregulated Mfn2 translate level compared to age-matched controls [75]. In an in- vitro neuronal cell model of AD, CU is reported to enhance Mfn2 expression and promotes protection against amyloid beta toxicity [133]. Further, Mfn2 overexpression in mice model is reported to ablate MPTP-mediated neurodegeneration in the substantia nigra of mice model [134 ]. Also, FOXO-mediated mitochondrial homeostasis is documented for neuronal and non-neuronal tissues [76,135 ]. Therefore, considering the evidences, insight from the current study suggests that CU-mediated differential modulation of Mfn2 driven by dFOXO underlies the ALSS neuroprotection. Like dFOXO (Fig 1b ) CU per se is also shown to enhance Mfn2 level only during HP ( Fig 3c ). Therefore, it is possible CU-mediated early life acting longevity enhancement, also underlies modulation of Bsk-dFOXO axis mediated Mfn2 modulation under the physiologic condition. To further understand mitochondrial respiratory capacity differential modulation ATP SynD was analysed which codes for a subunit-D of complex -V of respiratory chain. Onset of PD does not alter ATP SynD level during both the life stages ( Fig 3d ). Neurotoxicant PQ is not known for selectively inhibiting mitochondrial complexes [136], therefore the current observation corroborates the reports. CU intervention is shown to enhance ATP SynD level only during HP (Fig 3d). CU itself is an active mediator of ATP biogenesis [137 ], but enhancement of ATP SynD during in HP PD brain with CU intervention suggests that the respiratory capacity of neurons are elevated possibly due to overall betterment of mitochondrial health. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 48 4.4 CU-mediated ALSS neuroprotection in through HP specific modulation of CncC, GCLC, Prx 2540, Jafrac1 and Prx3 of the Phase II antioxidant defence system (ADS) CncC ( Drosophila orthologue of mammalian Nrf2) is a mediator of the phase II ADS which is responsible for the neutralization of the xenobiotics, toxins, and induced peroxide stress [78-80]. Further, some of the ADS components can be regulated by Bsk- dFOXO signalling [29,49,93 ]. In the present study, CncC expression in the PD brain was inhibited during HP and TP, whereas rescued with CU intervention only during HP ( Fig 4a). It was reported in in-vitro neuronal cells that exposure to sub-lethal PQ leads to depletion of Nrf2 which leads to impaired redox balance [81]. Further, CncC overexpression in the DAergic neuron of Drosophila ablates α -synuclein mediated toxicity, and neuronal death and enhances fly survival under harsh conditions [78] . Hence, CncC is highly necessary for optimal neuronal health and downregulation of the same is associated with the onset of PD. CU is a known modulator of Nrf2 in various neuronal disorders. CU and/or anti-cholinesterase combined formulation rescues diminished CncC expression and improves memory impairment in a young Drosophila model [138]. In a young mice model of focal ischemia and traumatic brain injury, CU intervention is demonstrated to alleviate neurodegenerative phenotype by rescuing diminished Nrf2 transcript level resulting in elevated level of downstream antioxidant gene expression [139]. Therefore, CU intervention promotes neuroprotection by rescuing the diminished level of CncC only during HP. Resuscitation of the CncC may contribute to the elevation of phase II ADS resulting in better neuronal defence during health phase but similar is not possible during TP. In the present study, GCLC expression was downregulated in the PD brain during HP, which was rescued upon CU intervention. But during TP there was a significant upregulation of GCLC in the PD brain which could not be altered with CU intervention preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 49 (Fig 4b ). GCLC expression is highly responsive to Nrf2/CncC signalling. Like CncC sublethal PQ exposure decreases GCLC level, leading to impaired redox balance [81] . Further, increased GCLC translate level due to CncC inducer treatment or DAergic CncC overexpression ablates α -synuclein mediated toxicity, and neuronal death in Drosophila [78]. Therefore, it is evident that CU mediated rescue of diminished GCLC driven by CncC underlies HP specific neuroprotection. However, GCLC upregulation in TP PD brain may be stress compensatory response, failure to rescue the altered level of the same by CU intervention suggest inability of CU to curb DAergic stress during later stages of life. Similarly, Jafrac1 expression in HP PD brain was downregulated which was rescued with CU intervention ( Fig 4f ). Lee et al, [93] also demonstrated that RNAi-mediated neuronal knockdown of Jafrac1 enhanced PQ-induced lethality in flies, whereas neuronal overexpression of the same reduced PQ-induced lethality. In DA neuronal cell culture and mice brains, it was reported that overexpression of Prx2 (A mammalian orthologue of Jafrac1) sequesters 6- OHDA-induced oxidative stress and neuronal death [140]. Further, Jafrac1 is also modulated my dFOXO [93], like CncC. Therefore, CU mediated HP-specific neuroprotection may underlie rescue of diminished Jafrac1 possibly driven by upstream dFOXO and/or CncC. In the PD brain of TP, expression of Jafrac1 is enhanced, which is further enhanced with CU intervention ( Fig 4 f). Considering the protective role of Jafrac1 it is possible that such upregulation in TP PD brain is an adult life stage-specific adaptive response to stress which is further enhanced by CU and is not driven by dFOXO and/or CncC. However, this enhanced Jafrac1 level may not protect from neurodegeneration during TP. Rescue of GCLC and Jafrac1 in HP suggest that reduced-GSH production is rescued and peroxide neutralization under the command of phase II mediator CncC and/or adaptive stress response mediator dFOXO. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 50 Interestingly the cross talk between adaptive stress response mediator dFOXO and phase II mediator CncC also extends to mitochondrial peroxiredoxins. Prx5 and Prx3 are two mitochondrial peroxiredoxins, where known modulator of Prx5 is CncC, Prx3 can also be modulated by dFOXO other than CncC. Prx5 product is ubiquitous to both mitochondrial and cytosol [36]. In the present study, Prx5 expression in the PD brain was inhibited during HP and TP which was rescued with CU intervention ( Fig 4h ). It was reported that Prx5 null flies show shortened life span by itself or with stress [97]. Further, global overexpression of the Prx5 increased resistance to oxidative stress and enhanced fly life span by 30% in normal conditions [97]. Therefore, it can be the inhibition of Prx5 during both the adult life stages is associated with the onset of PD, which was rescued with CU intervention. Yet, TP flies are not rescued with CU intervention, hence the ALSS neuroprotection is not through Prx5 modulation. Prx3 on the other hand codes for only mitochondria specific antioxidant [36] . Diminished level of the same under neurotoxicant insult leads to onset of PD in-vitro and neuronal over expression of the same in fly model enhances stress resistance particularly in middle age [96 ,141]. The current observation suggests like upstream dFOXO and CncC, diminished Prx3 level was rescued with CU intervention only during HP but not during transition phase ( Fig 4i). Therefore, insight suggests that diminished Prx3 level may lead to poor mitochondrial antioxidant capability in PD brain, which was corrected with during HP with CU intervention driven by dFOXO and/or CncC. In the population of good peroxiredoxins under the phase II ADS one acts as a double- edged sword. Prx 2540 or Prx 2540-1,2 other than having antioxidant capability has potent inflammatory capability [36]. Prx 2540 in Drosophila exist as two identical copies viz. Prx 2540-1 and Prx 2540-2 ( Prx 2540-1,2 ). In the present study, Prx 2540-1,2 expression was upregulated in the PD brain, whereas CU intervention normalized the preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 51 altered expression of the same only during HP ( Fig 4e). Due to its reported antioxidant capability [91], it can be postulated that upregulation of the same in PD brain is a compensatory response to stress. Upon inhibition of stress in HP the altered Prx 2540-1,2 level is rescued. But, it was also reported that PRDX6 (Mammalian orthologue of Prx 2540) transgenic mice show more DAergic neuronal loss, reduced tyrosine hydroxylase level and persistant inflammation upon MPTP exposure [142 ]. Therefore, in the current study, it is possible that in PD brain Prx 2540-1,2 upregulation may underlie DAergic neuronal degeneration and reduced tyrosine hydroxylase level as previously reported [143]. Owing to CU’s anti -inflammatory nature, intervention of the same inhibits Prx 2540-1,2 during HP and protects the neuron, but same is not possible in TP PD brain. Therefore, CU mediated ALSS neuroprotection underlies HP specific modulation of Prx 2540-1,2. Similarly, GCLM was upregulated in the PD brain during HP and TP, yet the altered level was rescued only during HP ( Fig 4c ). CncC downstream GCLM stabilizes GCLC- mediated GSH production [82]. GCLM is shown to promote neuroprotection and stress resistance [83, 144 ]. Therefore, the observed upregulation of the same in PD brain may be a stress compensatory response. CU could rescue the altered level of GCLM during HP but not during TP, suggesting CU can ablate some form of stress combated by GCLM only during HP, resulting in correction of the altered GCLM level under stress. GSTD1 of the phase II ADS is also under the control of Bsk signalling pathway [29]. It neutralizes xenobiotics and herbicides and shown to be upregulated in their presence in fly model [62,85,88]. Our observation corroborates the previous reports as in PD brain of both life stages GSTD1 level is enhanced, suggesting it is a general stress responder. CU intervention in PD brain does not alter the GSTD1 level, eluding that CU mediated ALSS neuroprotection is not through GSTD1. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 52 4.5 CU’s limitation in TP may underlie its failure to inhibit high iron intake signals in PD brain It was reported that metal (copper, heavy metal and iron) chelator protein-coding genes MtnA and Fer1HCH may respond to Bsk signalling in the Drosophila [29]. In the present study, MtnA and Fer1HCH expression in the PD brain was upregulated, while CU intervention could partially rescue the altered level during both the adult life stages ( Fig 5 a ,b). Mt-II (Vertebrate orthologue of fly MtnA) has previously been reported to be upregulated under neurotoxicant stress, traumatic injury in brain, which corroborates with our observation [100, 102]. MtnA are known for their role in heavy metal detoxification, anti-inflammatory and antioxidant role, that promotes neuroprotection against proteinopathic NDD like AD [145, 146]. Similarly, Fer1HCH is necessary for maintaining free iron chelation, preventing ROS generation in nervous system. Further, in Drosophila Fer1HCH is known to prevent axonal degeneration of axons and protect nervous system against ferroptosis [103, 104]. Therefore, upregulated MtnA and Fer1HCH in PD brain may be a stress compensatory response. Similarly high Fer1HCH expression is also observed in aging brain in Drosophila ( Fig S5b ), suggesting mechanism of aging and PD onset both leads to iron accumulation stress. CU intervention during both the adult life stages p artially rescue the upregulated MtnA and Fer1HCH levels, suggesting that CU intervention in the PD brain can somewhat prevent metal imbalance-associated stress. This action by CU may be due to its inherent chemistry, that enables it to perform as a metal chelating molecule [147]. However, this may not be enough to neuroprotection during later stages of life as observation suggests. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 53 Therefore, to further explore the reason behind inefficacy of CU we investigated the iron intake components of this metal homeostasis pathway. The enhanced IRP signalling

in the inhibition of Fer1HCH mediating iron chelation and simultaneously enhancing iron intake through activation of Tfr1 signalling [107]. In the present study, IRP-1A and IRP-1B expression in the PD brain was unaltered during HP, but expressions of the same were upregulated during TP. However, during TP CU intervention fully inhibited IRP-1A upregulation but IRP-1B upregulation was only partially rescued ( Fig 5 c,d). In PD and AD iron intake mediated by high IRP-IRE binding activity, has been a leading cause of the NDD [108,110]. Therefore, it is evident from the present study that in the PD brain upregulation of IRP-1A and IRP-1B during TP may contribute to the enhanced detriment in neurons. Further, natural aging also promoted extremely high IRP- 1A and significantly high IRP-1B expression in fly brains ( Fig S5 c, d). Thereby, suggesting cumulative iron intake signalling is enhanced upon PD onset during TP. CU intervention although inhibits IRP-1 A upregulation, it fails to completely rescue IRP-1B upregulation. Therefore, even though PD-associated IRP-1A upregulation is inhibited fully, the partial rescue of IRP-1B is not enough to curb the cumulative high IRP signalling intensity during TP. Hence, high iron intake and oxidative stress may still prevail during TP, highlighting one of the possible reasons for CU inefficacy during later adult life stages. 5. Conclusion Insights from the present study illustrate that possible players of CU-mediated HP- specific neuroprotection belong to stress-responsive Bsk signalling pathway, mitochondrial dynamics pathway and phase II ADS pathway. Further, these networks may have a potent cross-talk as different networks regulate similar downstream targets preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 54 and/or their action converges on the mitochondria ( Fig 6). The molecular players of CU mediated HP specific neuroprotection are (Fig 6)  Bsk-dFOXO stress response pathway: dFOXO, GADD45, Puc  Mitochondrial dynamics: Mfn2  Phase II ADS pathway: CncC, GCLC, Prx 2540 -1,2, Jafrac1, Prx3 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 55 Figure 6 : Cartoon depicts the involvement of molecular players in the HP-specific DAergic neuroprotective efficacy of CU. Insights reveal that CU-mediated ALSS DAergic neuroprotection underlies differential modulation of the molecular players belonging to the Bsk signalling pathway, mitochondrial dynamic pathway, and phase II ADS pathway. Failure to rescue the altered level of these players in TP PD brain suggests CU’s inefficacy during later stages of life. Further, CU-mediated inhibition of proteinopathic stress and some stress countered by Phase II ADS leads to the correction of altered levels of HSP68 and GCLM during HP only. Since, CU mediated HP specific neuroprotection promotes mitochondrial quality control in a dFOXO-Mfn2 dependent pathway and mitochondrial anti -oxidant capability in a CncC and/or dFOXO – Prx3 dependent pathway, it is concluded that mitocho ndrial health modulation may underlie life stage- specific neuroprotection. We would further like to emphasize that since CU’s inception as a phytomedicine/therapeutic molecule and despite its shortcomings as a therapeutic agent preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 56 in PD [10], still to these days CU has been proposed as a phytomedicine and one of the potential alternatives to currently available drugs when at least NDDs are concerned [148]. Perhaps a relook is necessary on how this potent nutraceutical be utilized to counter a late-onset diseases. Our study highlights that it is the natural aging associated molecular changes in neuro-biological pathways that renders therapeutic efficacy of CU as null in late-onset NDD such as PD. On the other hand, not therapeutic dosage, but dietary intervention of CU is a life span extender when fed at early life stages [115, 127], that is achieved through regulation of various molecular players. Perhaps if the molecular players can be sustained at a healthy regulatory level by dietary intervention of the nutraceutical, then the onset of the late-onset PD be prevented. Our lab is currently working in that direction. Further we are also working to sustain the observed brain- specific molecular players/pathways that confer DAergic neuroprotection during HP, in TP by adhering to different life course mediated feeding regimes. This understanding will help to modify existing therpapeutic strategies and also to develop novel therapeutic

for late-onset NDDs such as PD. Fundings: This research is supported by the Department of Biotechnology (DBT), India (R&D grant no. BT/405/NE/U-Excel/2013, 11-12 -2014) awarded to SCY .

Author Contributions: preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 57 Conceptualization:SCY Data curation:SCY, AD Formal analysis:AD Funding acquisition:SCY Investigation:AD, SCY Methodology:SCY Project administration:SCY Resources:SCY Software:AD, SCY Supervision:SCY Validation: AD, SCY Visualization: AD, SCY Writing - original draft:AD Writing - review & editing:SCY 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. Declarations Ethics Approval and Consent to Participate: Not applicable. Consent for Publication: Not applicable. Competing Interests: The authors declare no competing interests preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 58

1. Coppede F (2012) Genetics and epigenetics of Parkinson’s disease. Sci. World J. 2012:1–12. https://doi.org/10.1100/2012/489830. 2. Ramirez A, Heimbach A, Gründemann J, Stiller B, Hampshire D et al (2006) Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat. Genet. 38(10):1184 – 1191. https://doi.org/10.1038/ng1884. 3. Kalia LV, Lang AE (2015) Parkinson’s disease. Lancet 386(9996):896 –912. https://doi.org/1016/S0140-6736(14)61393-3. 4. De Miranda BR, Goldman SM, Miller GW, Greenamyre, JT, Dorsey ER (2022) Preventing Parkinson’s Disease: An Environmental Agenda. J. Parkinsons Dis . 12(1):45–68. https://doi.org/10.3233/JPD-212922. 5. Reichmann H (2015) Modern treatment in Parkinson’s disease, a personal approach. J. Neural. Transm.123(1):73 –80. https://doi.org/10.1007/s00702-015 - 1441-1. 6. Nakmode DD, Day CM, Song Y, Garg S (2023) The Management of Parkinson's Disease: An Overview of the Current Advancements in Drug Delivery Systems. Pharmaceutics. 15(1503). https://doi.org/10.3390/pharmaceutics15051503. 7. Hickey P, Stacy M (2011) Available and emerging treatments for Parkinson’s disease: A review. Drug. Des. Devel. The. 5 :241–254. https://doi.org/10.2147/DDDT.S11836. 8. Rascol O, Payoux P, Ory F, Ferreira JJ, Brefel-Courbon C et al (2003)

of current Parkinson’s disease therapy. Ann. Neurol . 53(3). https://doi.org/10.1002/ana.10513. 9. Kunnumakkara AB, Hegde M, Parama D, Girisa S, Kumar A et al (2023) Role of Turmeric and Curcumin in Prevention and Treatment of Chronic Diseases: Lessons Learned from Clinical Trials. ACS. Pharmacol. Transl. Sci. 6(4):447 – 518. https://doi.org//10.1021/acsptsci.2c00012. 10. Ghodsi H, Rahimi HR, Aghili SM, Saberi A, Shoeibi A (2022) Evaluation of curcumin as add- on therapy in patients with Parkinson’s disease: A pilot randomized, triple-blind, placebo-controlled trial. Clin. Neurol. Neurosurg. 218:107300. https://doi.org/10.1016/j.clineuro.2022.107300. 11. Ayajuddin M, Phom L, Koza Z, Modi P, Das A et al (2022) Adult health and transition stage-specific rotenone mediated Drosophila model of Parkinson’s disease: Impact on Late-onset Neurodegenerative Disease Models. Front. Mol. Neurosci. 15:896183. https://doi.org/10.3389/fnmol.2022.896183. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 59 12. Phom L, Achumi B, Alone DP, Muralidhara, Yenisetti SC (2014) Curcumin’s neuroprotective efficacy in Drosophila model of idiopathic Parkinson’s disease is phase specific: Implication of its therapeutic effectiveness. Rejuvenation Res. 17:481–489. https://doi.org/10.1089/rej.2014.1591. 13. Arking R, Novoseltseva J, Hwangbo DS, Novoseltsev V, Lane M (2002) Different Age-Specific Demographic Profiles Are Generated in the Same Normal -Lived Drosophila Strain by Different Longevity Stimuli. J. Gerontol. 57(11):390-398. https://doi.org/10.1093/gerona/57.11.b390. 14. Pletcher SD, Macdonald SJ, Marguerie R, Certa U, Stearns, SC et al (2002) Genome-Wide Transcript Profiles in Aging and Calorically Restricted Drosophila melanogaster. Curr. Biol.12(9):712 –723 . https://doi.org/10.1016/S0960- 9822(02)00808-4. 15. Yang J, Huang T, Petralia F, Long Q, Zhang B et al (2015) Synchronized age- related gene expression changes across multiple tissues in human and the link to complex diseases. Sci. Rep. 5. https://doi.org/10.1038/srep15145. 16. Kumar A, Gibbs JR, Beilina A, Dillman A, Kumaran R et al (2013) Age- associated changes in gene expression in human brain and isolated neurons. Neurobiol. Aging 34(4):1199 –1209. https://doi.org/10.1016/j.neurobiolaging.2012.10.021. 17. Ayajuddin M, Chaurasia R, Das A, Modi P, Phom L et al (2023) Fluorescence microscopy-based sensitive method to quantify dopaminergic neurodegeneration in a Drosophila model of Parkinson’s disease. Front. Neurosci. 17:1158858. https://doi.org/10.3389/fnins.2023.1158858 18. Niveditha S, Shivanandappa T (2022) Potentiation of paraquat toxicity by inhibition of the antioxidant defenses and protective effect of the natural antioxidant, 4-hydroxyisopthalic acid in Drosophila melanogaster. Comparative Biochemistry and Physiology Part C: Toxicology and Pharmacology, 259, 109399. https://doi.org/10.1016/j.cbpc.2022.109399. 19. Ortega-Arellano HF, Jimenez-Del-Rio M, Velez-Pardo C (2021) Melatonin increases life span, restores the locomotor activity, and reduces lipid p eroxidation (LPO) in transgenic k nockdown parkin Drosophila melanogaster exposed to paraquat or p araquat/iron. Neurotox. Res. 39(5):1551 –1563. https://doi.org/10.1007/s12640-021-00397-z. 20. Niveditha S, Ramesh SR, Shivanandappa T (2018) Correction to: Paraquat- induced movement disorder in relation to oxidative stress-mediated neurodegeneration in the brain of Drosophila melanogaster. Neurochem. Res. 43(2):515– 516. https://doi.org/10.1007/s11064-017-2458-7. 21. Ravi SK, Narasingappa RB, Joshi CG, Girish TK, Vincent B (2018) Neuroprotective effects of Cassia tora against paraquat induced neurodegeneration: Relevance for Parkinson’s disease. Nat. Prod. Res. 32(12):1476– 1480. https://doi.org/10.1080/14786419.2017.1353504. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 60 22. Jhonsa DJ, Badgujar LB, Sutariya BK, Saraf MN, Cicero AFG, Saraf M (2016) Neuroprotective effect of flavonoids against paraquat induced oxidative stress and neurotoxicity in Drosophila melanogaster. Curr. Top. Nutraceutical Res. 14(4):283– 294. 23. Phom L (2018) Understanding neurodegeneration and rescuing pathology associated with Parkinsons disease in Drosophila model. Dissertation, Nagaland University, Lumami. http://hdl.handle.net/10603/327054. 24. See WZC, Naidu R, Tang KS (2023) Paraquat and Parkinson's disease: The molecular crosstalk of upstream signal transduction pathways leading to apoptosis. Curr Neuropharmacol. https://doi.org/10.2174/1570159X21666230126161524. 25. Gan T, Fan L, Zhao L, Misra M, Liu M et al (2021) Jnk signaling in Drosophila aging and longevity. Int. J. Mol. Sci. 22:9649. https://doi.org/10.3390/ijms22179649. 26. Wang L, Davis SS, Borch Jensen M, Rodriguez-Fernandez IA, Apaydin C et al. (2019). JNK modifies neuronal metabolism to promote proteostasis and longevity. Aging Cell 18:e12849. https://doi.org/10.1111/acel.12849. 27. Biteau B, Karpac J, Hwangbo DS Jasper H (2011) Regulation of Drosophila lifespan by JNK signaling. Exp. Gerontol. 46 :349–354. https://doi.org/10.1016/j.exger.2010.11.003. 28. Wang MC, Bohmann D, Jasper H (2005) JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. Cell 121 :115–125. https://doi.org/10.1016/j.cell.2005.02.030. 29. Wang MC, Bohmann D, Jasper H (2003) JNK Signaling Confers tolerance to o xidative stress and extends lifespan in Drosophila. Dev. Cel l 5(5):811–816. https://doi.org/10.1016/S1534-5807(03)00323-x. 30. Perluigi, M., Di Domenico, F., and Butterfield, D.A. (2015). mTOR signalling in aging and neurodegeneration: At the crossroad between metabolism dysfunction and impairment of autophagy. Neurobiol Dis. 84, 39-49. https://doi.org/10.1016/j.nbd.2015.03.014. 31. Partridge L, Alic N, Bjedov I, Piper MDW (2011) Ageing in Drosophila: The role of the insulin/Igf and TOR signalling network. Exp. Gerontol. 46 :376-381. https://doi.org/10.1016/j.exger.2010.09.003. 32. Wong M (2013) Mammalian target of rapamycin (mTOR) pathways in neurological diseases. Biomed. J. 36 :40–50. https://doi.org/10.4103/2319- 4170.110365. 33. Stern M, McNew JA (2021) A transition to degeneration triggered by oxidative stress in degenerative disorders. Mol. Psychiatry 26 :736–746. https://doi.org/10.1038/s41380-020-00943-9. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 61 34. Stern M (2017) Evidence that a mitochondrial death spiral underlies antagonistic pleiotropy. Aging Cell. 16:435-443. https://doi.org/10.1111/acel.12579. 35. Li PA, Hou X, Hao S (2017). Mitochondrial biogenesis in neurodegeneration. J. Neurosci. Res. 95:2025-2029. https://doi.org/10.1002/jnr.24042. 36. Detienne G. De, Haes W, Mergan L, Edwards SL, Temmerman L, Van Bael S (2018) Beyond ROS clearance: Peroxiredoxins in stress signalling and aging. Ageing Res. Rev. 44:33-48. https://doi.org/10.1016/j.arr.2018.03.005. 37. Zhang M, An C, Gao Y, Leak RK, Chen J, Zhang F (2013) Emerging roles of Nrf2 and phase II antioxidant enzymes in neuroprotection. Prog. Neurobiol. 100:30-47. https://doi.org/10.1016/j.pneurobio.2012.09.003. 38. Singh N, Haldar S, Tripathi AK, Horback K, Wong J et al. (2014). Brain Iron Homeostasis: From molecular mechanisms to clinical significance and therapeutic opportunities. ARS. 20:1324 –1363. https://doi.org/10.1089/ars.2012.4931. 39. Hidalgo J, Penkowa M, Giralt M, Carrasco J, Molinero A (2002) Metallothionein Expression and Oxidative Stress in the Brain In Protein sensors and reactive oxygen species. Methods in Enzymology 348:238-249. 40. Luckinbill LS, Arking R, Clare MJ, Cirocco WC Buck SA (1984) Selection for delayed senescence in Drosophila melanogaster. Evolution 38 :996–1003. https://doi.org/10.1111/j.1558-5646.1984.tb00369.x 41. Das A, Chaurasia R, Phom L, Modi P, Yenisetti SC (2021). Quantitative Real Time PCR (qRT-PCR) for Gene Expression Analysis. In: Lakhotia SC and Ranganath HA (ed) Experiments with Drosophila for Biology Courses, Indian Academy of Sciences, pp 351-359. ISBN: 978-81-950664-2-1. 42. Livak KJ, Schmittgen TD (2001). Analysis of Relative Gene Expression Data Using Real Time Quantitative PCR and the 2 -∆∆Ct Method. Methods 25 :402-408. https://doi.org/10.1006/meth.2001.1262. 43. Tas D, Stickley L, Miozzo F, Koch R, Loncle N, Sabado, V (2018) Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons. PLoS Genet. 14. https://doi.org/10.1371/journal.pgen.1007271. 44. Koh H, Kim H, Kim MJ, Park J, Lee HJ, Chung J (2012) Silent information regulator 2 (Sir2) and forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant. JBC. 287:12750 –12758. https://doi.org/10.1074/jbc.M111.337907. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 62 45. Zhu Z, Reiser G (2018) The small heat shock proteins, especially HspB4 and HspB5 are promising protectants in neurodegenerative diseases. Neurochem. Int. 115:69-79. https://doi.org/10.1016/j.neuint.2018.02.006. 46. Tettweiler G, Miron M, Jenkins M, Sonenberg N, Lasko PF (2005) Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP. Genes Dev. 19 :1840–1843. https://doi.org/10.1101/gad.1311805. 47. Keshavarz R, Bakhshinejad B, Babashah S, Baghi N, Sadeghizadeh M (2016). Dendrosomal nanocurcumin and p53 overexpression synergistically trigger apoptosis in glioblastoma cells. Iran. J. Basic Med. Sci. 19 :1353–1362. https://doi.org/10.22038/ijbms.2016.7923. 48. Moskalev A, Plyusnina E, Shaposhnikov M, Shilova L, Kazachenok A, Zhavoronkov A (2012) The role of D-GADD45 in oxidative, thermal and genotoxic stress resistance. Cell Cycle 11:4222 – 4241. https://doi.org/10.4161/cc.22545. 49. Van der Vos KE, Coffer PJ (2011) The extending network of FOXO transcriptional target genes. ARS. 14 :579–592. https://doi.org/10.1089/ars.2010.3419. 50. Plyusnina EN, Shaposhnikov MV, and Moskalev, A.A. (2011). Increase of Drosophila melanogaster lifespan due to D-GADD45 overexpression in the nervous system. Biogerontology. 12, 211 –226. https://doi.org/10.1007/s10522- 010-9311-6. 51. Kojima S, Mayumi-Matsuda K, Suzuki H, Sakata T (1999) Molecular cloning of rat GADD45ϒ, gene induction and its role during neuronal cell death. FEBS Lett. 446 :313-7. https://doi.org/10.1016/s0014-5793(99)00234-3 52. Gonda RL, Garlena RA, Stronach B (2012) Drosophila heat shock response requires the jnk pathway and phosphorylation of mixed lineage kinase at a conserved serine-proline motif. PLoS ONE 7(7 ). https://doi.org/10.1371/journal.pone.0042369. 53. Huang H, Lu-Bo Y, Haddad GG (2014) A Drosophila ABC Transporter Regulates Lifespan. PLoS Genet. 10:10048 44. https://doi.org/10.1371/journal.pgen. 54. Tare M, Modi RM, Nainaparampil JJ, Puli OR, Bedi S et al (2011) Activation of JNK signaling mediates amyloid-ß -dependent cell death. PLoS ONE 6. https://doi.org/10.1371/journal.pone.0024361. 55. McEwen DG, Peifer M (2005) Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis. Development 132 :3935–3946. https://doi.org/10.1242/dev.01949. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 63 56. Li J, Du W, Maynard S, Andreassen PR, Pang Q (2010) Oxidative stress-specific interaction between FANCD2 and FOXO3a. Blood 115:1545 –1548. https://doi.org/10.1182/blood-2009-07-234385. 57. Van der Horst A, Burgering BMT (2007) Stressing the role of FoxO proteins in lifespan and disease. Nat. Rev. Mol. Cell Biol. 8 :440–450. https://doi.org/10.1038/nrm2190. 58. Maier CM, Chan PH (2002) Role of superoxide dismutases in oxidative damage and neurodegenerative disorders. Neuroscientist 8 :323-34. https://doi.org/10.1177/107385840200800408. 59. Arking R, Burde V, Graves K, Hari R, Feldman E et al. (2000a). Identical longevity phenotypes are characterized by different patterns of gene expression and oxidative damage. Exp. Gerontol. 35 :353-373. https://doi.org/10.1016/s0531-5565(00)00096-6. 60. Arking R, Burde V, Graves K, Hari R, Feldman E, Zeevi A, et al (2000b) Forward and reverse selection for longevity in Drosophila is characterized by alteration of antioxidant gene expression and oxidative damage patterns. Exp . Gerontol. 35:167-185. https://doi.org/10.1016/s0531-5565(99)00094-7. 61. Arking R (2001) Gene Expression and Regulation in the Extended Longevity Phenotypes of Drosophila. Ann. N. Y. Acad . Sci. 928:157 -167. https://doi.org/10.1111/j.1749-6632.2001.tb05645.x 62. Park JH, Jung JW, Ahn YJ, Kwon HW (2012) Neuroprotective properties of phytochemicals against paraquat-induced oxidative stress and neurotoxicity in Drosophila melanogaster . Pestic. Biochem. Phys. 104(2):118 –125. https://doi.org/10.1016/j.pestbp.2012.07.006. 63. Hirano Y, Kuriyama Y, Miyashita T, Horiuchi J, Saitoe M (2012) Reactive oxygen species are not involved in the onset of age-related memory impairment in Drosophila. G2B. 11:79 –86. https://doi.org/10.1111/j.1601- 183X.2011.00748.x. 64. Nässel DR, Liu Y, Luo J (2015) Insulin/IGF signalling and its regulation in Drosophila. Gen. Comp. Endocrinol. 221:255 -266. https://doi.org/10.1016/j.ygcen.2014.11.021. 65. Grönke S, Clarke DF, Broughton S, Andrews TD, Partridge L (2010) Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet. 6(2). https://doi.org/10.1371/journal.pgen.1000857. 66. Broughton SJ, Piper MDW, Ikeya T, Bass TM, Jacobson J et al (2005). Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands. Proc. Natl. Acad . Sci. 102:3105 -3110. https://doi.org/10.1073/pnas.0405775102. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 64 67. Holzenberger M, Kappeler L, Filho CDM (2004) IGF-1 signalling and aging. Exp. Gerontol. 39:1761–1764. https://doi.org/10.1016/j.exger.2004.08.017. 68. Slack C, Alic N, Foley A, Cabecinha M, Hoddinott MP, Partridge L (2015) The Ras-Erk -ETS-Signaling Pathway Is a Drug Target for Longevity. Cell 162:72–83. https://doi.org/10.1016/j.cell.2015.06.023. 69. Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H et al. (2001). Extension of Life-Span by Loss of CHICO, a Drosophila Insulin Receptor Substrate Protein. Science 292:104. https://doi.org/10.1126/science.1057991. 70. Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S (2004). Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr. Biol. 14:885–890. https://doi.org/10.1016/j.cub.2004.03.059. 71. Asahara SI, Inoue H, Watanabe H, Kido Y (2022) Roles of mTOR in the Regulation of Pancreatic β -Cell Mass and Insulin Secretion. Biomolecules 12 :614. https://doi.org/10.3390/biom12050614. 72. Morita M, Gravel SP, Hulea L, Larsson O, Pollak M et al (2015) mTOR coordinates protein synthesis, mitochondrial activity. Cell Cycle 14:473 -480. https://doi.org/10.4161/15384101.2014.991572. 73. Icreverzi A, Flor De La Cruz A, Van Voorhies WA, Edgar BA (2012). Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress. Cell Cycle 11:554 –568. https://doi.org/10.4161/cc.11.3.19062. 74. de Oliveira Souza A, Couto-Lima CA, Rosa Machado MC, Espreafico EM, Pinheiro Ramos RG, Alberici LC (2017) Protective action of Omega-3 on paraquat intoxication in Drosophila melanogaster. J. Toxicol. Environ. Health. A. 80:1050-1063. https://doi.org/10.1080/15287394.2017.1357345. 75. Zhao F, Wang W, Wang C, Siedlak SL, Fujioka H, Tang B (2017) Mfn2 protects dopaminergic neurons exposed to paraquat both in vitro and in vivo: Implications for idiopathic Parkinson’s disease. Bioch im. Biophys. Acta. Mol. Basis. Dis. 1863:1359-1370. https://doi.org/10.1016/j.bbadis.2017.02.016. 76. Hagenbuchner J, Ausserlechner MJ (2013) Mitochondria and FOXO3: Breath or die. Front. physiol. 4:147. https://doi.org/10.3389/fphys.2013.00147. 77. Jonckheere AI, Smeitink JAM, Rodenburg RJT (2012). Mitochondrial ATP synthase: Architecture, function and pathology. J. Inherit. Metab. Dis. 35:211 - 225. https://doi.org/10.1007/s10545-011-9382-9. 78. Wang B, Liu Q, Shan H, Xia C, Liu Z (2015) Nrf2 inducer and cncC overexpression attenuates neurodegeneration due to α -synuclein in Drosophila. Biochem. Cell Biol. 93:351–358. https://doi.org/10.1139/bcb-2015-0015. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 65 79. Tufekci KU, Civi Bayin E, Genc S, Genc K (2011) The Nrf2/ARE pathway: A promising target to counteract mitochondrial dysfunction in Parkinson’s disease. Parkinsons Dis. 2011:314082. https://doi.org/10.4061/2011/314082. 80. Sykiotis GP, Bohmann D (2010) Stress- activated cap’n’collar transcription factors in aging and human disease. Sci. Signal. 3:re3. https://doi.org/10.1126/scisignal.3112re3. 81. Narasimhan M, Riar AK, Rathinam ML, Vedpathak D, Henderson G, Mahimainathan L (2014) Hydrogen peroxide responsive miR153 targets Nrf2/ARE cytoprotection in paraquat induced dopaminergic neurotoxicity. Toxicol. Lett. 228:179-191. https://doi.org/10.1016/j.toxlet.2014.05.020. 82. Franklin CC, Backos DS, Mohar I, White CC, Forman HJ, Kavanagh TJ (2009). Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase. Mol. Aspects Med. 30:86 -98. https://doi.org/10.1016/j.mam.2008.08.009. 83. Cole TB, Giordano G, Co AL, Mohar I, Kavanagh, TJ, Costa LG (2011). Behavioral characterization of GCLM -knockout mice, a model for enhanced susceptibility to oxidative stress. J. Toxicol. 2011:157687. https://doi.org/10.1155/2011/157687. 84. Trinh K, Moore K, Wes PD, Muchowski PJ, Dey J et al. (2008) Induction of the phase II detoxification pathway suppresses neuron loss in Drosophila models of Parkinson's disease. J. Neurosci. 28:465-472. https://doi.org/10.1523/JNEUROSCI.4778-07.2008. 85. Maitra U, Harding T, Liang Q, Ciesla, L (2021) GardeninA confers neuroprotection against environmental toxin in a Drosophila model of Parkinson’s disease. Commun. Biol. 4:162. https://doi.org/10.1038/s42003-021- 01685 -2. 86. Zemanová M, Stašková T, Kodrík D (2016) Role of adipokinetic hormone and adenosine in the anti-stress response in Drosophila melanogaster. J. Insect. Physiol. 91-92:39–47. https://doi.org/10.1016/j.jinsphys.2016.06.010. 87. Smeyne M, Smeynen RJ (2013) Glutathione metabolism and Parkinson’s disease. Free Radic. Biol. Med. 62:13 -25. https://doi.org/10.1016/j.freeradbiomed.2013.05.001. 88. Vrailas-Mortimer A, Gomez R, Dowse H, Sanyal S (2012) A survey of the protective effects of some commercially available antioxidant supplements in genetically and chemically induced models of oxidative stress in Drosophila melanogaster. Exp. Gerontol. 47:712 –722. https://doi.org/10.1016/j.exger.2012.06.016. 89. Drolet NA (2019). Peroxiredoxin 6 and Phospholipids in Alzheimer's Disease. Dissertation. Dedman College of Humanities and Sciences, US. https://scholar.smu.edu/hum_sci_biologicalsciences_etds/4. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 66 90. Ma Q (2013) Role of Nrf2 in oxidative stress and toxicity. Annu. Rev. Pharmacol. Toxicol. 53:401 -426. https://doi.org/10.1146/annurev-pharmtox- 011112-140320. 91. Radyuk SN, Klichko VI, Spinola B, Sohal RS, Orr WC (2001) The peroxiredoxin gene family in Drosophila melanogaster. Free Radic. Biol. Med. 31 :1090-1100. https://doi.org/10.1016/S0891-5849(01)00692-X 92. DeGennaro M, Hurd TR, Siekhaus DE, Biteau B, Jasper H, Lehmann R (2011) Peroxiredoxin Stabilization of DE-Cadherin Promotes Primordial Germ Cell Adhesion. Dev. Cell. 20:233 –243. https://doi.org/10.1016/j.devcel.2010.12.007. 93. Lee KS, Iijima-Ando K, Iijima K, Lee WJ, Lee JH, Yu K et al (2009) JNK/FOXO-mediated neuronal expression of fly homologue of peroxiredoxin II reduces oxidative stress and extends life span. JBC. 284:29454 –29461. https://doi.org/10.1074/jbc.M109.028027. 94. Radyuk SN, Klichko VI, Michalak K, Orr WC (2013) The effect of peroxiredoxin 4 on fly physiology is a complex interplay of antioxidant and signalling functions. FASEB J. 27:1426 –1438. https://doi.org/10.1096/fj.12-214106 95. Odnokoz O, Nakatsuka K, Klichko VI, Nguyen J, Solis LC et al (2017) Mitochondrial peroxiredoxins are essential in regulating the relationship between Drosophila immunity and aging. Biochim. Biophys. Acta - Mol. Basis Dis. 1863 :68–80. https://doi.org/10.1016/j.bbadis.2016.10.017. 96. Radyuk SN, Rebrin I, Klichko VI, Sohal BH, Michalak K, Benes J (2010) Mitochondrial peroxiredoxins are critical for the maintenance of redox state and the survival of adult Drosophila. Free Radic. Biol. Med. 4 9:1892–1902. https://doi.org/10.1016/j.freeradbiomed.2010.09.014. 97. Radyuk SN, Michalak K, Klichko VI, Benes J, Rebrin I et al (2009) Peroxiredoxin 5 confers protection against oxidative stress and apoptosis and also promotes longevity in Drosophila. Biochem J. 419 :437–445. https://doi.org/10.1042/BJ20082003. 98. Yiwen W, Xiaohan T, Chunfeng Z, Xiaoyu Y, Yaodong M Huanhuan Q (2022). Genetics of metallothioneins in Drosophila melanogaster. Chemosphere 288 :132562. https://doi.org/10.1016/j.chemosphere.2021.132562. 99. Ramnarine TJS, Glaser-Schmitt A, Catalán A, Parsch J (2019) Population genetic and functional analysis of a cis-regulatory polymorphism in the Drosophila melanogaster Metallothionein a gene. Genes 10. https://doi.org/10.3390/genes10020147. 100. Najib NHM, Yahaya MF, Das S, Teoh SL (2021) The effects of metallothionein in paraquat-induced Parkinson disease model of zebrafish. Int. J. Neurosci.133:822-833. https://doi.org/10.1080/00207454.2021.1990916. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 67 101. Sharma S, Ebadi M (2014). Significance of metallothioneins in aging brain. Neurochem. Int. 65:40-48. https://doi.org/10.1016/j.neuint.2013.12.009. 102. Penkowa M (2006). Metallothionein I + II expression and roles during neuropathology in the CNS. Dan. Med. Bull. 53:105-121. 103. Mukherjee C, Kling T, Russo B, Miebach K, Kess E et al (2020) Oligodendrocytes Provide Antioxidant Defense Function for Neurons by Secreting Ferritin Heavy Chain. Cell Metab. 32:259 -272.e10. https://doi.org/10.1016/j.cmet.2020.05.019. 104. González-Morales N, Mendoza-Ortíz MÁ, Blowes LM, Missirlis F, Riesgo- Escovar JR (2015). Ferritin is required in multiple tissues during Drosophila melanogaster development. PLoS ONE 10(7 ). https://doi.org/10.1371/journal.pone.0133499. 105. Kaur D, Yantiri F, Rajagopalan S, Kumar J, Mo JQ et al (2003) Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson's disease. Neuron 37:899 -909. https://doi.org/10.1016/s0896-6273(03)00126-0. 106. Jellinger K (1999) The role of iron in neurodegeneration: prospects for pharmacotherapy of Parkinson’s disease. Drugs Aging 14 :115–140. https://doi.org/10.2165/00002512-199914020-00004. 107. Wilkinson N, Pantopoulos K (2014) The IRP/IRE system in vivo: Insights from mouse models. Front. Pharmacol. 5:176. https://doi.org/10.3389/fphar.2014.00176. 108. Cho HH, Cahill CM, Vanderburg CR, Scherzer CR, Wang B et al (2010) Selective translational control of the Alzheimer amyloid precursor protein transcript by iron regulatory protein-1. JBC. 285:31217 –31232. https://doi.org/10.1074/jbc.M110.149161. 109. Lind MI, Missirlis F, Melefors Ö, Uhrigshardt H, Kirby K et al (2006) Of two cytosolic aconitases expressed in Drosophila, only one functions as an iron- regulatory protein. JBC. 281:18707 –18714. https://doi.org/10.1074/jbc.M603354200. 110. Faucheux BA, Martin ME, Beaumont C, Hunot S, Hauw JJ et al (2002) Lack of up -regulation of ferritin is associated with sustained iron regulatory protein-1 binding activity in the substantia nigra of patients with Parkinson’s disease. J. Neuro chem. 83:320–330. https://doi.org/10.1046/j.1471-4159.2002.01118.x. 111. Srivastav S, Fatima M, Mondal AC (2018) Bacopa monnieri alleviates paraquat induced toxicity in Drosophila by inhibiting jnk mediated apoptosis through improved mitochondrial function and redox stabilization. Neurochem. Int. 121:98-107. https://doi.org/10.1016/j.neuint.2018.10.001 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 68 112. Xiang L, Nakamura Y, Lim Y, Yamasaki Y, Kurokawa ‐ Nose Y et al (2011) Tetrahydrocurcumin extends life span and inhibits the oxidative stress response by regulating the FOXO forkhead transcription factor. Aging 3:1098 -1109. https://doi.org/10.18632/aging.100396. 113. Senchuk MM, Van Raamsdonk JM, Moore DJ (2021) Multiple genetic pathways regulating lifespan extension are neuroprotective in a G2019S LRRK2 nematode model of Parkinson's disease. Neurobiol. Dis. 151 :105267. https://doi.org/10.1016/j.nbd.2021.105267. 114. Arking R (2015) Independent chemical regulation of health and senescent spans in Drosophila. IRD. 59:28–32. https://doi.org/10.1080/07924259.2014.978028. 115. Soh JW, Marowsky N, Nichols TJ, Rahman AM, Miah T et al (2013) Curcumin is an early-acting stage-specific inducer of extended functional longevity in Drosophila. Exp. Gerontol. 48(2):229– 239. https://doi.org/10.1016/j.exger.2012.09.007. 116. McLaughlin CN, Broihier HT (2018) Keeping Neurons Young and Foxy: FoxOs Promote Neuronal Plasticity. Trends Genet. 34:65 -78. https://doi.org/10.1016/j.tig.2017.10.002. 117. Maitra U, Scaglione MN, Chtarbanova S, O’Donnell JM (2019) Innate immune responses to paraquat exposure in a Drosophila model of Parkinson’s disease. Sci. Rep. 9:12714. https://doi.org/10.1038/s41598-019-48977-6. 118. Eroglu B, Jin X, Deane S, Öztürk B, Ross OA et al. (2022) Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death. Cell Mol. Life. Sci. 79:198. https://doi.org/10.1007/s00018-022-04162-z. 119. Renton AEM (2010) Gene expression and genetic analyses in Parkinson’s disease with and without dementia. Dissertation. University College, London https://discovery.ucl.ac.uk/516147/1/516147.pdf. 120. Morimoto RI, Tissieres A, Georgopoulos C (1994) The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, US. 121. Rallis A, Navarro JA, Rass M, Hu A, Birman S, Schneuwly S et al (2020) Hedgehog Signaling Modulates Glial Proteostasis and Lifespan. Cell Rep. 30 :2627-2643.e5. https://doi.org/10.1016/j.celrep.2020.02.006. 122. Slack C, Giannakou ME, Foley A, Goss M, Partridge L (2011) dFOXO- independent effects of reduced insulin-like signaling in Drosophila. Aging Cell 10 :735–748. https://doi.org/10.1111/j.1474-9726.2011.00707.x. 123. Karpac J, Hull-Thompson J, Falleur M, Jasper H (2009) JNK signaling in insulin- producing cells is required for adaptive responses to stress in Drosophila. Aging Cell. 8:288– 295. https://doi.org/10.1111/j.1474-9726.2009.00476.x. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 69 124. Sanz FJ, Solana-Manrique C, Lilao-Garzón J, Brito-Casillas Y, Muñoz-Descalzo S, Paricio N (2022) Exploring the link between Parkinson’s disease and type 2 diabetes mellitus in Drosophila. FASEB J. 36:e22432. https://doi.org/10.1096/fj.202200286R. 125. Grönke S, Clarke DF, Broughton S, Andrews TD, Partridge L (2010) Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet. 6(2). https://doi.org/10.1371/journal.pgen.1000857. 126. Wills J, Credle J, Oaks AW, Duka V, Lee JH, Jones J et al (2012) Paraquat, but not maneb, induces synucleinopathy and tauopathy in striata of mice through inhibition of proteasomal and autophagic pathways. PLoS One 7:e30745. https://doi.org/10.1371/journal.pone.0030745. 127. Lee KS, Lee BS, Semnani S, Avanesian A, Um CY et al (2010) Curcumin extends life span, improves health span, and modulates the expression of age- associated aging genes in Drosophila melanogaster . Rejuvenation Res. 13:561– 570. https://doi.org/10.1089/rej.2010.1031. 128. Wang YL, Ju B, Zhang YZ, Yin HL, Liu YJ et al (2017) Protective effect of curcumin against oxidative stress- induced injury in rats with Parkinson’s disease through the Wnt/β -catenin signaling pathway. Cell Physiol. Biochem. 43(6):2226–2241. https://doi.org/10.1159/000484302. 129. Piao Y, Kim HG, Oh MS, Pak, YK (2012). Overexpression of TFAM, NRF-1 and myr-AKT protects the MPP +-induced mitochondrial dysfunctions in neuronal cells. Biochim. Biophys. Acta. 1820:577 -585. https://doi.org/10.1016/j.bbagen.2011.08.007. 130. Gureev AP, Shaforostova EA, Popov, VN (2019) Regulation of mitochondrial biogenesis as a way for active longevity: Interaction between the Nrf2 and PGC- 1α signalling pathways. Front. genet. 10 :435. https://doi.org/10.3389/fgene.2019.00435. 131. Chin D, Hagl S, Hoehn A, Huebbe P, Pallauf K et al (2014) Adenosine triphosphate concentrations are higher in the brain of APOE3- compared to APOE4-targeted replacement mice and can be modulated by curcumin. Genes Nutr. 9:397. https://doi.org/10.1007/s12263-014-0397-3. 132. Icreverzi A, de la Cruz AFA, Walker DW, Edgar BA (2015) Changes in neuronal CycD/Cdk4 activity affect aging, neurodegeneration, and oxidative stress. Aging Cell 14:896– 906. https://doi.org/10.1111/acel.12376. 133. Reddy PH, Manczak M, Yin X, Grady MC, Mitchell A, Kandimalla R (2016) Protective effects of a natural product, curcumin, against amyloid β induced mitochondrial and synaptic toxicities in Alzheimer’s disease. JIM. 64 : 1220– 1234. https://doi.org/10.1136/jim-2016-000240. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 70 134. Zhao F, Austria Q, Wang W, Zhu X (2021) Mfn2 overexpression attenuates mptp neurotoxicity in vivo. Int. J. Mol. Sci. 22:1 –14. https://doi.org/10.3390/ijms22020601 135. Cheng Z (2022) Foxo transcription factors in mitochondrial homeostasis. Biochem. J. 479:525-536. https://doi.org/10.1042/BCJ20210777. 136. Richardson JR, Quan Y, Sherer TB, Greenamyre JT, Miller GW (2005) Paraquat neurotoxicity is distinct from that of MPTP and Rotenone. Toxicol. Sci. 88:193 – 201. https://doi.org/10.1093/toxsci/kfi304. 137. Gibellini L, Bianchini E, De Biasi S, Nasi M, Cossarizza A, Pinti M (2015). Natural Compounds Modulating Mitochondrial Functions. eCAM. 2015:527209. https://doi.org/10.1155/2015/527209. 138. Ogunsuyi OB, Aro OP, Oboh G, Olagoke OC (2022) Curcumin improves the ability of donepezil to ameliorate memory impairment in Drosophila melanogaster: involvement of cholinergic and cnc/Nrf2-redox systems. Drug Chem. Toxicol. 7:1-9. https://doi.org/10.1080/01480545.2022.2119995. 139. Dong W, Yang B, Wang L, Li B, Guo X et al (2018) Curcumin plays neuroprotective roles against traumatic brain injury partly via Nrf2 signaling. TAPI. 346:28–36. https://doi.org/10.1016/j.taap.2018.03.020. 140. Hu X, Weng Z, Chu CT, Zhang L, Cao G et al (2011) Peroxiredoxin-2 protects against 6-hydroxydopamine-induced dopaminergic neurodegeneration via attenuation of the Apoptosis Signal-Regulating Kinase (ASK1) signalling cascade. Journal of Neuroscience 31:247 –261. https://doi.org/10.1523/JNEUROSCI.4589-10.2011. 141. Rajput C, Sarkar A, Singh MP (2021) Involvement of Peroxiredoxin- 3, Thioredoxin-2, and Protein Deglycase-1 in Cypermethrin-Induced Parkinsonism. Mol. Neurobiol. 58 : 4745–4757. https://doi.org/10.1007/s12035-021-02456-0. 142. Yun HM, Choi DY, Oh KW, Hong JT (2015) PRDX6 Exacerbates Dopaminergic Neurodegeneration in a MPTP Mouse Model of Parkinson’s Disease. Mol. Neurobiol. 52:422–431. https://doi.org//10.1007/s12035-014-8885-4. 143. Das A (2022) Mechanistic insights into the neuroprotective efficacy of curcumin in a Drosophila model of P arkinson’s Disease. Dissertation, Nagaland Universuty, Lumami. http://nuir.inflibnet.ac.in:8080/jspui/handle/123456789/570. 144. Liang LP, Kavanagh TJ, Patel M (2013). Glutathione deficiency in Gclm null mice results in complex I inhibition and dopamine depletion following paraquat administration. Toxicol. Sci. 134:366–373. https://doi.org/10.1093/toxsci/kft112. 145. Juarez-Rebollar D, Rios C, Nava-Ruiz C (2017) Metallothionein in brain disorders. Oxid. Med. Cell. Longev. 2017:5828056. https://doi.org/10.1155/2017/5828056. preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 71 146. Xu W, Xu Q, Cheng H (2017) The efficacy and pharmacological mechanism of Zn7MT3 to protect against Alzheimer’s disease. Sci . Rep. 7:13763. https://doi.org/10.1038/s41598-017-12800-x. 147. El Nebrisi E (2021) Neuroprotective activities of curcumin in Parkinson’s disease: A review of the literature. Int. J. Mol. Sci. 22:11248. https://doi.org/10.3390/ijms222011248. 148. Madhubala D, Patra A, Khan MR, Mukherjee AK (2024) Phytomedicine for neurodegenerative diseases: The road ahead. PTR, https://doi.org/10.1002/ptr.8192. Supplementary Figures preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 72 Figure S1 : Brain-specific aging associated changes in expression of molecular players involved in Bsk signalling pathway. Natural aging in brain inhibits Bsk-dFOXO stress response axis, as expression level of Bsk and dFOXO was inhibited in TP brain as compared to that of HP (a,b). However, dFOXO downstream l(2)efl expression was unaltered, whereas 4e-bp and GADD45 expression were upregulated in TP brain as compared to HP (c,d,e). Similarly Bsk downstream HSP68 expression was also enhanced, whereas Bsk downstream Puc expression was inhibited in TP brain as compared to HP (f,g). dFOXO downstream antioxidant gene SOD1 and CAT remain unaltered, whereas SOD2 expression was inhibited in TP brain as compared to HP (h,i,j). Significance was drawn by analysing the data of minimum three replicates with unpaired t-Test. (*p<0.05; **p<0.01; ***p<0.001; NS: Not significant) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 73 Figure S2: Brain-specific aging associated changes in expression of molecular players involved in IIS- dTOR signalling pathway. Natural aging in the brain may enhance cumulative IIS signalling as expression level ILP2 was enhanced, whereas ILP3 was downregulated and ILP5 remained unaltered in TP brain as compared to that of HP (a,b,c). Similarly, ILP downstream Chico level was also upregulated (d). However, unlike IIS, the downstream dTOR-RS6K1 signalling cascade may be repressed with natural aging as dTOR and RS6K1 level was inhibited in the TP brain as compared to that of HP (e,f). Significance was drawn by analysing the data of minimum three replicates with unpaired t-Test . (*p<0.05; **p<0.01; ***p<0.001; NS: Not significant) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 74 Figure S3: Brain-specific aging associated changes in expression of molecular players involved in mitochondrial dynamics. Natural aging in brain inhibits mito-biogenesis capacity as Ewg and TFAM expression level were inhibited in the TP brain as compared to that of HP (a,b). However, mito-quality control is enhanced with natural aging as Mfn2 level was upregulated in the TP brain as compared to that of HP (c). Natural aging diminishes respiratory capacity in the brain as ATP SynD was diminished in the TP brain as compared to that of HP (d). Significance was drawn by analysing the data of minimum three replicates with unpaired t-Test. (*p<0.05; **p<0.01; ***p<0.001; NS: Not significant) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 75 Figure S4: Brain-specific aging associated changes in expression of molecular players involved in phase II ADS. Natural aging in the brain upregulates phase II ADS mediator CncC (a). However, CncC downstream GCLC and GCLM were downregulated in the TP brain, suggesting a decline in GSH synthesis with aging (b,c). Further, GSTD1, Prx 2540-1,2 , Jafrac2, Prx5 and Prx3 were upregulated in TP brain, whereas Jafrac1 was downregulated (d,e,f,g,h,i). Significance was drawn by analysing the data of minimum three replicates with unpaired t-Test. (*p<0.05; **p<0.01; ***p<0.001; NS: Not significant) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 76 Figure S5: Brain-specific aging associated changes in expression of molecular players involved in metal homeostasis. Natural aging in the brain did not alter MtnA expression (a). But with aging iron accumulation in the brain may be enhanced and in response to that Fer1HCH was upregulated in TP brain (b). Similarly in the TP brain iron uptake was also enhanced as IRP-1A and IRP-1B expression was upregulated (c,d). Significance was drawn by analysing the data of minimum three replicates with unpaired t-Test. ( *p<0.05; **p<0.01; ***p<0.001; NS: Not significant) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 77 Melting curves for gene-specific primers Bsk dFOXO l(2)efl 4e-bp GADD45 HSP68 Puc SOD1 SOD2 CAT preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 78 ILP2 ILP3 ILP5 Chico dTOR RS6K1 Ewg TFAM Mfn2 ATP synD Prx 2540-1,2 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint 79 CncC GCLC GCLM GSTD1 Jafrac1 Jafrac2 Prx5 Prx3 MtnA Fer1HCH IRP-1A IRP-1B Rp49 preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted January 18, 2025. ; https://doi.org/10.1101/2025.01.17.633690doi: bioRxiv preprint