Phenolic monoterpene Bakuchiol ameliorates age related Parkinsonism

doi:10.21203/rs.3.rs-4652207/v1

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Phenolic monoterpene Bakuchiol ameliorates age related Parkinsonism

https://doi.org/10.21203/rs.3.rs-4652207/v1

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The phenomenon of aging comes with various spontaneous physiological changes including structural, functional, molecular and cellular. whereas, deterioration from the normal physiology brings chronic changes associated with several neurodegenerative disorders. Neurodegenerative diseases related to aging are the most concerned topic due to their devitalizing conditions with lack of therapy options that simultaneously affects the economic and social status. In our study we illustrated the longevity and anti-parkinsonism activity of Bakuchiol (BAK), a phytomolecule from Psoralea corylifolia. Our result showed BAK elevate mean life span up to 38% along with the enhanced survival rate under stress. Additionally, BAK reduces ROS level via boosting anti-oxidant level through upregulating expression of anti- oxidative genes sod-3 and gst-4. Remarkably, BAK exhibits neuroprotective effects by attenuating α-synuclein levels. These findings suggest that, in addition to its antioxidant and lifespan-extending properties, BAK positively regulates neurological health. In summary, our study establishes the potential curative effects of BAK on age-related neurodegenerative disorders, positioning it as a promising nootropic drug in modern medicine with broader implications for age-related neurodegeneration.

Bakuchiol

neurological disease

ROS

longevity

In the current scenario, increased life expectancy is the leading cause of diseases associated with aging. Aging is a major risk aspect for diverse neurological anarchy including Alzheimer's disease (AD) [1], Parkinson's disease (PD) [2], cerebrovascular disease (CVD) [3] and Amyotrophic lateral sclerosis (ALS). The major theories which are interlinked have been reported to regulate aging are free radical induced oxidative stress and mitochondrial dysfunction and biogenesis [4]. Mitochondrial dysfunction and unbalanced ROS production has been recommended as the major causes in the pathogenesis of these neurological dystrophys. These neurodegenerative diseases described by the accumulation of toxic proteins including α-synuclein [5], amyloid-beta and neurofibrillary tangles [6] related with progressive deterioration of neurons. Pathologically neurological diseases are portrayed by degeneration of neurons which leads to the deficit of neurotransmitter viz. acetylcholine and dopamine. Recent reports suggested that mitochondrial dysfunction, oxidative stress and misfolded protein have a key factors in the pathogenesis of neurological disorders.

Antioxidants viz. vitamin C [7], vitamin E [8] and flavonoids [9] have been reported to protect against oxidative stress by nullifying free radicals thus expanding healthy ageing. Presently, permanent cure for neurological disorders is still unknown, the medications for symptomatic relief are provided only along with their side-effects. Considering the side effects while using synthetic drugs, the idea for developing new medicine is diverted toward ayurveda (natural products) in the present scenario. Large varieties of herbs have been known from ages for their medicinal properties in curing numbers of diseases. Several plants have been reported for their anti-aging and neuroprotective activities like, Withania somnifera [10], Mucunapruriens [11], Curcuma longa and Bacopa monnieri [12]. In the current study, with the reported efficacy of phytomolecules, we undertook the present study to elucidate the effect of bakuchiol (BAK) on lifespan and Parkinsons Disease. Earlier, we have reported the role of BAK in the improvement of cholinergic function and oxidative stress tolerance in C. elegans [13].

2.1. Maintenance of cultures and chemicals

Bakuchiol was obtained from phytocompound, Karnataka, India. All the strains of C. elegans utilized in current study were procured from Caenorhabditis Genetic Center (CGC), university of Minnesota (www.cgc.cbs.umn.edu). Maintenance of the worm culture were done on freshly prepared nematode growth medium plates with lawn of live OP50 strain of E .coli as a food source at 20°C [14]. The chemicals used in current study were of highly standard quality from Sigma and Merck companies. The strains utilized in present study were wild type Bristol N2, transgenic strain NL5901 (Punc-54::α-synuclein:: YFP + unc-119), CF1553 (sod-3::gfp), CL2166 (gst-4::gfp).

2.2. Life span assay

Life span assay was performed to illustrate the efficacy of BAK in improving longevity on wild type N2 and transgenic strain TK22 (mev-1). Isolation of eggs from gravid worm’s body was done by alkaline hypochlorite method [15]. The isolated synchronized eggs of N2 were transferred on freshly prepared NGM plates with different doses (6.25µM, 12.5µM, 25µM, 37.5µM and 50µM) and control plate seeded with live OP50, followed by incubation period of 5 days at 20°C. Healthy adult worms were transferred on freshly prepared plates having 50µM of 5-fluoro-2- deoxyuridine (FUdR) to stop progeny development [16] with treatment and OP50 in triplicate group. Worms were transferred to new treatment plates on regular intervals to avoid any type of contamination and risk of progeny mixing in the population. Survival of worms was observed on daily basis by tapping and touching with poking lashes. Death of all worms was marked as termination of experiment. The lifespan assay with the strains TK22 was performed with same procedure as discussed at effective concentration 37.5µM of BAK. Statistical analysis of data was done by applying kaplan-Meier test under GraphPad prism software. The experiment was performed in three different biological replicate groups.

2.3. Pharengeal Pumping

Changes in worms standard lifespan and shifts in their eating patterns have been linked. Dietary restriction (DR)-like effects that prolong lifespan have been linked to the decline in feeding rate. To examine how exposure to bakuchiol impacts feeding behavior, an alteration in the pharyngeal pumping rate was assessed. The pharyngeal pumping rate is the rate at which the pharyngeal bulb of a worm contracts and relaxes once every minute, and a drop in pharyngeal pumping is linked to DR in worms. Day 5 and day 10 worms (n = 20) pretreated with bakuchiol had their pharyngeal pumping rate measured at regular intervals of 20 seconds for one minute at room temperature. A total of three sessions of the test were carried out.

2.4. Brood Size

The longevity trait has been linked to a reduction in descendants production. Therefore, total progeny production complying with exposure to the various treatments was recorded in order to analyse the impacts of BAK. According to the established protocol, the assay was carried out. The worms ages were coordinated. Individually selected L4 stage worms were subsequently placed on new plates with previously spotted treatment compound and vehicle control. Every day until the reproduction ceased the worms were individually relocated to a new plate that had been previously marked with BAK and DMSO control. At the L2 or L3 stage, the offspring of each worm under observation were counted. The experiment was performed thrice.

2.5. Intestinal lipofuscin assay

Lipofuscin is an autofluoroscent granules containing residual lipid that aggregates proportionally to aging and known as biological hall mark of aging. Briefly, Age synchronized population of eggs were allowed to hatch on treatment and control plates. On 5th day of adulthood randomly selected worms were placed onto a glass slide having 20µl of M9 buffer and 4µl Sodium Azide (From 100mM) and a cover slip placed over it. The auto-fluorescent was observed and captured using DAPI filter (with excitation at 365 nm and emission at 420 nm) under fluorescence microscope (Leica, DMI3000) at 20X. The experiment was carried out three times and data quantification was done by using Image J software.

2.6. Juglone survival assay

Antioxidant mechanism involve in the balancing of oxidative stress by quenching reactive oxygen species (ROS) thus improves aging associated deterioration [17]. To investigate impact of BAK on enhancing in vivo antioxidant properties in wild type worms, juglone (5-Hydroxy-1,4-napthoquinone, Sigma-Aldrich) an intracellular ROS generator was introduced. The age-synchronized worms were treated with 37.5µM BAK and control. The treated and control healthy adult day 2 worms were moved to 24-well plate containing 300µl liquid NGM and oxidative stress was generated by addition of Juglone (250 µM) per well and incubation of the plate at 20°C. Further, scoring was done for the number of live worms till 50% of total worms in control group were found dead. The data was represented as percentage of survival (Mean ± S.E.) by using unpaired t-test in GraphPad prism software version 5.01. The experiment performed independently three times.

2.7. Gene Reporter assay of stress response genes SOD-3 & GST-4

Antioxidant enzymes are the chief defense mechanism to balance the ROS production by catalyzing them into stable molecules. To determine the effect of BAK on antioxidant enzymes, transgenic strain CF1553 (sod-3::gfp) and CL2166 (gst-4::gfp) were used. Age synchronized embryos were transferred on treated and control plates and incubated at 20°C for 5 days. Randomly picked healthy hermaphrodites nematodes in 20µl M9 buffer along with 4µl sodium azide and mounted a cover slip over it. The fluorescent intensity was captured at 20X using Leica fluorescent microscope (DMI 3000B, Germany) with excitation-emission at wavelength 365nm-420nm.

2.8. Lipid detection assay by Nile red staining method

Adipogenesis during aging has severe impact on aging processes and responsible for the aetiology of chronic disorders [18]. For the investigation of BAK action on lipid accumulation during aging Nile Red, a lipophilc dye was used to stain and measure fat droplets in worm’s body. The eggs of wild type worms were transferred on both treated and control plates pre seeded with nile red (5 mg/ml stock dissolved in acetone) with OP50 in the ratio of 250:1 (OP50:Nile red). The plates were incubated at 20°C for 5 days. Worms were washed off thrice using M9 buffer and 100mM Sodium Azide was added to anesthetize the worms. Randomly selected worms placed onto the glass slide for capturing the images under rhodamine filter using DMI 3000B leica fluorescent microscope (Weltzar, Germany) at 20X. The quantification of images was done by using Image J software and analysis was done using version 5.01 of Graph Pad prism software. The experiment was performed in thrice independent biological replicates and data represented as Mean ± S.E.

2.9. Acridine orange staining assay

Acridine orange dye provides a simple examination procedure to segregate among live, apoptotic and necrotic cells [19]. In current experimentation, worms treated with BAK and control were washed thrice by using M9 buffer. 100µl of acridine orange dye (5mg/ml in DDH₂O) per sample was added to 1.5ml centrifuge tube containing worm suspension along with 100µl M9 buffer. Afterward, the tubes were incubated on a rotator shaker for 30 minutes together with covering the tubes with silver foil (for avoiding the reaction of acridine orange with light). After 30 minutes the worms were washed with M9 buffer thrice and worm pallet was added to liquid OP50 for 45 min and incubated at 20^OC to remove the stained bacteria present inside the gut. The worms were washed off again and anesthetized by adding 4µl of sodium azide (100 mM). The anesthetized worms were transferred on glass slide, fixed by cover slip and visualized under a fluorescent microscope utilizing DAPI channel (Leica, Wetzlar, Germany) at 20X. The quantification of fluorescent intensity was measured by using imageJ software and statistical analysis was performed under GraphPad Prism software.

2.10. α-Synuclein accumulation level

The elevated α-synuclein level in the substantia nigra region of brain is known for major pathogenicity and hallmark for PD. To illustrate the efficacy of BAK on α-synuclein aggregation, transgenic strain NL5901 (Punc-54:: α-synuclein:: YFP + unc-119) of C. elegans was utilized. Worms of transgenic strain NL5901 were grown on live OP50 with BAK at different doses 6.25 µM, 12.5 µM, 37.5 µM, 25 µM, and 50 µM including a control group at 20^OC. After 5 days, synchronized adult hermaphrodites were washed with M9 buffer thrice and transferred on Glass slide with 4 µl sodium azide utilized for immobilization and covered with a glass cover slip. Imaging of live worms was performed under a GFP channel (with excitation at range of 365 nm and emission at range of 420 nm) under the DMI 3000 B (Leica, Wetzlar, Germany) fluorescence microscope. The fluorescence intensity was evaluated utilizing software ImageJ and analysis was done by Graphpad Prism software respect of Mean ± S.E. The ImageJ estimates the fluorescence intensity over the picked region (kept same for each subject) and provides numbers regarding relative fluorescence units.

2.11. 1-Nonanol Repulsion Assay

Neurotransmitter Dopamine (DA) regulates motor functions of C. elegans and the deviation from the normal DA levels affects the response time of the worms towards attractant or repellant [20]. To measure the efficacy of BAK on Dopamine level, 1-Nonanol, a chemo-repellent was used on wild type and transgenic NL5901 worms. L1 stage Wild type and NL5901 worms were treated with BAK and control. Close to the head of a forward moving worm poking lash dipped in 1-nonanol was placed. Worms shows a quick reaction against the chemo repellent and reverses the moving path; time taken by worms for repulsion was recorded. Further analysis was done by unpaired t-test in Graph Pad Prism software of version 5.01.

2.12. Body Bend

Basal ganglia regulates the motor movement and activates dopaminergic neurons whereas during PD, impairment in basal ganglia causes loss in body rhythm and movement [21]. For the study of BAK role on basal ganglia in wild type N2 and transgenic strain NL5901, body bend movement assay was examined. To investigate the body bends, adult day 2 worms (treated/untreated) were placed onto a glass slide with 20µl of buffer M9 and examined for the quantity of body bends in an interim of 20s. A body bend was included as a change in the position of the part of nematode forming upside down S- Shape. A body bend was counted as a change in the direction of the part of the nematodes corresponding to the posterior bulb of the pharynx along the Y-axis, assuming that the nematode was traveling along the X-axis. Data was analyzed by applying unpaired t-test in GraphPad prism software (version 5.01).

2.13. Head Thrash

PD is characterized by dysfunction of dopaminergic neurons which impairs the motor neuron and thus causes posture imbalance and tremor [21]. Head thrash assay was performed to check the impact of BAK on health of motor neurons of wild type and NL5901 strain of C. elegans. To measure the head thrash, healthy adult treated and control nematodes were washed thrice with M9 buffer. Nematodes from sample were place onto a glass slide containing a drop of M9 buffer (approx. 20 µl). After 1 min recuperation period, the head thrash were measured and noted per 20 second for each treatment. A thrash was characterized as a movement of head in to and fro direction. The comparative analysis of data was done by applying unpaired t-test in GraphPad prism software.

2.14. Isolation of RNA, synthesis of RNA and cDNA and quantification of mRNA expression

For the total RNA extraction, synchronized embryos were grown onto the treatment and control plates at 22ºC for 5 days. Adult day 2 worms were washed off with M9 buffer to remove bacteria. To isolate RNA, RNAzol method was used as reported in earlier studies. The synthesis of cDNA from RNA was done by the following protocol as described in thermofisher cDNA synthesis kit. For detection in the respective mRNA expression, quantification was done by performing qRT-PCR for lrk-1, lagr-1, pdr-1, pink-1, ymel-1, and ubc-12 and housekeeping gene gpd-1, using SyberGreen method. The quantification in mRNA expression was obtained by calculating comparative C_T method (_T).

3.1. BAK extended the life span

BAK extend the mean life span of wild type N2 strain of C. elegans. BAK treated worms shows significant increase in mean life span at 12.5µM (17.37 ± 0.011), 25µM (17.44 ± 0.01327), 37.5µM (18.22 ± 0.011) in comparison to control (13.2 ± 0.008) whereas, it was close to control at 6.25µM (17.11 ± 0.002) and 50µM (16.94 ± 0.0214) (Fig. 1B, Table. 1). Maximum elevation in mean life span observed at 37.5µM about to 38% in wild type strain of C. elegans, as illustrate in Fig. 1C. On comparison with specific dose at 37.5µM (14.7 ± 0.0366) in mutant strain TK-22 shows 17% increase in life span when compared to control (12.56 ± 0.0255).

3.2. BAK curtailed the oxidative stress in C. elegans

Generation of reactive oxygen species exerts oxidative stress which promotes various age-related disorders. Juglone survival assay was accomplished to detect the consequences of BAK on oxidative stress tolerance against Juglone (a source of reactive oxygen species (ROS) within the cell) exposure [22]. In our study we noticed that there was a considerable spike in survival percent to 92% in BAK treated wild type N2 worms (96.00 ± 0.577, p < 0.0001) compared to control group (50.01 ± 2.942) (Fig. 2A). For further confirmation of anti-oxidant activity in BAK, life span of transgenic strain TK22 (mev-1) was performed compared to control worms, the outcome reveals an increase in survival of up to 17.03%. (Fig. 2B). Together the results indicated that BAK possesses significant oxidative stress tolerance activity in C. elegans.

3.3. BAK demolishes the intracellular ROS level in nematodes

H2DCF-DA staining method used for detecting the presence of intracellular ROS level inside the worm’s body [23]. We observed that wild type worms treated with BAK at 37.5µM decline the relative ROS level effectively up to 23% (p < 0.01) in comparision to untreated group. Moreover, in TK22 11% (p < 0.05) compared to the corresponding control, BAK-treated worms showed a decrease in intracellular ROS levels. The comparison between the relative ROS level in wild type N2 and TK22 strains of C. elegans were depicted in Fig. 2C.

3.4. BAK elevates expression of SOD-3 and GST-4 in C. elegans

Anti-oxidative gene sod-3 (superoxide dismutase) encodes for mitochondrial anti-oxidative enzymes and gst-4 (glutathione S-transferase) is significant stage-II detoxification enzymes known for balancing intracellular ROS level [24]. The BAK at 37.5µM significantly enhanced the SOD-3 expression by (1.389 ± 0.043, p < 0.001) 38% in contrast to control worms (1.001 ± 0.066) (Fig. 2D-F) whereas expression level of GST-4 was enhanced by (1.428 ± 0.070, p < 0.0001) 42% in comparison with control worms (1.000 ± 0.012) (Fig. 3A-C). The significant impact of BAK on stress and lifespan may be attributable to improved expression levels of SOD-3 and GST-4, according to data. BAK balances the ROS level by enhancing the synthesis of anti-oxidant enzymes.

3.5. BAK demolishes the lipid content in wild type model of C. elegans

According to recent studies Parkinson’s disease are caused by α- synuclein as well as anomalous lipid levels as a result of modifications at molecular level [5]. In wild type N2 worms treated with BAK, the lipid level was measured.Worms of control group displayed high lipid level (1.000 ± 0.035) in relation to decreased level of up to 43% upon treatment with 37.5µM BAK (0.578 ± 0.002, p < 0.0001) as presented in graph (Fig. 3D-F). This indicates BAK help in delaying the process of aging with declination in neurological disorders by lowering down the lipid level.

3.6. BAK curtails apoptosis

Mitochondrial oxidative stress is associated with aging that promotes violations of apoptotic pathway. In the current examination the impact of BAK on apoptosis was detected utilizing acridine orange staining assay. At 37.5µM dose of BAK wild type worms shows significant increase in cellular health to 29% as compared to control group as shown in Fig. 3G-I.

3.7. BAK attenuates α-Synuclein aggregation level in transgenic strain NL5901

Parkinson’s disease is age dependent disease accompanied by accumulation of α-synuclein in form of lewy body in substantia nigra pars compacta region of the brain [25–26]. Using the transgenic strain N5901, this study sought to show how BAK affects α-synuclein aggregation. We found that BAK treatment significantly reduces the α-synuclein level at 6.25µM (0.762 ± 0.040, p < 0.0001), 12.5µM (0.735 ± 0.006, p < 0.0001), 25µM (0.689 ± 0.013, p < 0.0001), 37.5µM (0.662 ± 0.014, p < 0.0001) and 50µM (0.693 ± 0.043, p < 0.0001) in contrast with control group (1.000 ± 0.002). Among all doses highest level of reduction in α-synuclein level was observed at 37.5µM to 34%. Data suggests that BAK, potentially attenuated α-synuclein level in diseased conditions. The graphs and microphotographs shown in Fig. 4A-F indicate the effect of BAK on α-synuclein in diseased worm NL5901.

3.8. Enhancement of dopamine level in C.elegans by BAK

Degeneration of dopaminergic neurons results in the decreased dopamine levels in the organisms. Chemo-repulsive behavior against 1-nonanol is associated with dopamine level, higher the dopamine level indicates quick response and vice versa [20]. According to our findings, In both wild type and NL5901 strains, the control group had the best mean repulsion time, but worms treated with BAK showed a substantial reduction in mean repulsion time in wild type (42%, p < 0.0001) and transgenic strain NL5901 (32%, p < 0.0001). The graph in Fig. 4G represents the comparative analysis between wild type and in diseased condition.

3.9. BAK boost locomotary action in C. elegans

Numerous types of body motions, including head thrashing and body bending can be used to examine nematode motion, which is designated as a end point behavioral parameter and associated to neuronal health. BAK treatment raised the thrashing of treated wild type worms by 23% while non-significant effect was observed in transgenic NL5901 worms in comparison to control (Fig. 4H). Additionally, BAK enhanced body bend in wild type worms by 27% and 29% in diseased condition in contrast with their respective controls (Fig. 4I). The data suggests the efficacy of BAK in boosting the health of motor neurons.

The customary arrangement of medication has used herbs for treating different human disorders. The side effects of medications from synthetic source have forced again towards the utilization of herbal medication for restoring age related issues in human beings. Although herbal remedies based on medicinal plants have existed for centuries, scientific validation and testing are required for creating natural meds. Numerous examinations have demonstrated that Plant-derived molecules and extracts can reduce age-related disorders and expand life expectancy. As a result, the current study aimed to determine the anti-aging potential of Bakuchiol (BAK) and its antioxidant response in relation to Parkinsonism. Therefore, we assessed impact of BAK on life expectancy and stress responses utilizing C. elegans animal model. Current examination shows non-toxic effect of BAK in range of 5µM-1mM and recommends that 37.5µM dose maximally extend mean life expectancy by 38% in C. elegans as evident through lifespan assay. Various endpoint parameters related to aging were known in which lipofuscin aggregation was most discussed one. Lipofuscin is a lipoprotein aggregates occurs because of covalent cross linking and accumulated in aged cells which lead to the deleterious effects over the span of life. Therefore, to justify our study of BAK in delaying aging, we perform lipofuscin aggregation assay. The BAK treatment lessened lipofuscin accumulation in worm by 44%. The augmentation of oxidative stress because of raised ROS level or thermal-induced stress is one of the main contributing variables to many age-related pathologies including Alzheimer's disease, Parkinson's disease and cardiovascular disorders with age movement. Therefore, we deciphered impacts of BAK in understanding its role on stress generation because of high temperature and production of ROS in animal model C. elegans. High temperature denatures the proteins and other biomolecules and enhances in formation of ROS inside the cells, promoting the process of aging as well as related disorders. Various chaperons mediated protein inside the cell helps in mitigating and balancing the thermal induced stress and protect the cell and tissue from damaging and maintain the proper signaling. As BAK increase the life span; therefore, it was assumed that it might be having some positive response in regulating thermal stress. To justify our hypothesis, we performed thermotolerance assay and expectedly we found survival rate after heat shock in treated worm increased to 95%. This shows BAK possess thermotolerance activity and can be helpful in delaying aging and related disorders.

In consideration with previous reports that the anti-oxidant activity of licorice on C. elegans by using Juglone [27], we investigate the impact of BAK on in vivo ROS level by performing Juglone survival assay. Our study reveals that BAK possesses antioxidant activity as it reduces the induced ROS level to 92%. It was reported in several research article that transgenic strain TK22 [mev-1(kn-1)] deficient in cytochrome b (mev-1) protein that helps in respiratory chain complex-II in integral mitochondrial membrane experiences shorter life span because of oxidative stress [28] For the justification of our reported data that BAK possess anti-oxidant activity, we perform life span assay with TK22 mutant strain. Expectedly, we observed extended mean life span about to 17% in BAK treated mutant mev-1, showing that it is proficient to diminish oxidative stress in TK22. BAK effectively regulates the oxidative stress and hence enhances the life span. Synchronization between the generation of ROS and its defense mechanism is necessary in order to maintain proper balance between the rate of production of oxidative stress to the rate of antioxidant scavenging activity [29]. Any disturbance in these phenomena lead to oxidative stress. So, for balancing the generated ROS, anti-oxidative enzymes play key roles. Studies have reported the role of sod-3 and gst-4 helps in reducing the ROS level in different animal models including C. elegans, mice and Drosophila melanogaster [30]. In this way, we evaluated GST-4 and SOD-3 expression, two stress-responsive antioxidant enzymes tagged with GFP in the transgenic C. elegans. The BAK introduction elevated the expression of gst-4 and sod-3 by 42% and 38% respectively. The outcomes suggested that BAK advances the anti-oxidant enzyme mechanism in C. elegans.

Such remarkable reduction in ROS level and elevation in mev-1 mean life span confirms efficiency of BAK in providing anti-oxidant activity and enhancing the expression of enzymes creates a path for mechanistic aspects in C. elegans. We have reviewed from previous reports that the negative effect of ROS on damaging the healthy cells and generating various related disorders. Therefore, we were interested in inspecting the role of BAK on viability of cells. As it efficiently reduces the ROS level in our previous experiments, we expected its activity in improving cellular health. Remarkably, BAK improves the cellular health significantly to 29% evident through acridine orange staining. The oxidation of biomolecules (lipids and proteins) because of ROS and other stress in living cells are responsible for causing deleterious effects on organism’s health. It was reported that lipid oxidation leads to formation of lipid peroxidation, that also act as second messenger for stress and responsible in causing immune-senescence, sarcopenia and osteoporosis [31–32] and also involved in promoting cancer. Lipid peroxidation and its role in aging and related disorders also reported in previous studies [33]. In the same view, we measured the efficacy of BAK against lipid deposition inside the cell. The result suggested that BAK impressively reduces the neutral lipid level approximately to 43% in C. elegans. Till now, we observed that BAK effectively delayed aging and help in elevating anti-oxidant activities and relatively lower the oxidative stress which protects oxidation of biomolecules too.

Crosstalk between aging and neurodegenerative disorder are always the topic of concern, especially when it comes to PD. Previous investigation draws the relation between the etiology of PD with ageing and oxidative stress. With this we hypothesised, as BAK helps in improving lifespan and demolishing the oxidative stress therefore to some extend it also had some effect on PD associated parameters too. In previous reports the role of ROS in enhancing the accumulation of toxic α-synuclein protein was reported. The results from our study justified our hypothesis as we found significant alleviation in α-synuclein aggregation level to 34%. Toxic protein α-synuclein accumulation leads to decrease dopamine level with degeneration of dopaminergic neurons [34]. Another investigation reported the repulsion due to 1-nonanol directly relates the dopamine level in C. elegans [20] so, introduction of BAK showed quick and stronger response indicating higher dopamine level in both wild type approximately to 42% and mutant strain NL5901 to 32%. Previous reports suggested that locomotary behavior is dependable on dopamine levels in worms [35], our investigation reveals both wild type and mutant strain NL5901 produced higher level of body movement and head thrash thus indicating positive effect of BAK on dopaminergic neurons thus help in reducing the aetiology of PD. To validate our findings, qPCR analyses were conducted on lrk-1, the human ortholog being LRRK1 which encodes for synaptic vesicle transmission and plays a role in dopamine regulation. The substantial upregulation of lrk-1 signifies the impact of BAK-induced exacerbation of movement dysfunction related to Parkinson's disease (PD). Additionally, the increased expression of ymel-1 (a mitochondrial protease), analogous to the presenilin-associated metalloprotease in humans and recognized as a suppressor of α-synuclein inclusion formation, suggests BAK's involvement in curtailing α-synuclein aggregates. The mitochondrial serine/threonine kinase, pink-1, facilitates mitophagy through mitochondrial trafficking termination via phosphorylation. The increased mRNA expression level of pink-1 confirmed that pink-1 is responsible for BAK-induced normal functioning of mitochondria. BAK was found to significantly reduce neutral lipid levels, with an elevated ceramide level, the smallest sphingolipids, leading to decreased triglyceride levels. To further support our conclusions, we investigated the mRNA expression level of lagr-1, an ortholog of human CESR1 and LAG1 in yeast, which is a sphingolipid synthetase involved in sphingolipid synthesis. The increased expression of lagr-1 establishes BAK's role in diminishing lipid peroxidation to alleviate PD pathology. Notably, pdr-1, an ortholog of human parkin (PARK2), a crucial gene in managing proteotoxic stress through the ubiquitin proteasome system, physically associates and collaborates with a conserved degradation machinery to mediate ubiquitin conjugation, leading to the degradation of α-synuclein protein. Furthermore, mutations in pdr-1 are known to increase susceptibility to mitochondrial complex I inhibitors. The involvement of the ubiquitin proteasome system was also observed through the upregulation of ubc-12, encoding an essential ubiquitin-conjugating enzyme in this system. Increased expression level of ubc-12 and pdr-1 results that BAK utilizes the pdr-1 mediated ubiquitin proteasome system to counter α-synuclein aggregation.

Altogether, the study shows that BAK can be utilized against age associated neurodegenerative disease PD. The study exhibits tremendous potential in curtailing the oxidative stress and ROS level, lipid accumulation while increasing the expression of anti-oxidative enzymes activity, improving the life span, modulation of neurotransmitters like dopamine along with the mitigation of misfolded toxic proteins viz. α-synuclein aggregation. Therefore, findings of this study can be used for future research in higher animal model for gaining mechanistic insights of BAK and the study will be useful therapeutic interventions that unravel as promising geroprotective and nootropic drug.

BAK Bakuchiol

PD Parkinson's disease

GFP Green fluorescent protein

YFP Yellow fluorescent protein

ROS Reactive oxygen species

DMSO Dimethyl sulfoxide

NGM Nematode growth medium

H2DCF-DA 2', 7'-dichlorodihydrofluorescein diacetate

FUDR 5-fluorodeoxyuridine

DA Dopamine

Data Availability

The data used to support the findings of this study are available in the article. Raw data are available on request from the corresponding author upon reasonable request.

Acknowledgments

The authors acknowledge the Research and Development Office of Integral University, Lucknow, for facilitating the communication of the manuscript through the provision of the manuscript communication number (IU/R & D/2022-MCN0001698). Additionally, the authors would like to express their heartfelt gratitude to the Director, CSIR-CIMAP Lucknow.

Funding

This research was supported by CSIR-CIMAP Lucknow.

Competing Interests

The authors declare no competing interests.

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Table.1. Effect of BAK on lifespan of C. elegans

Genotype	Treatment	Mean	±S.E.	Sample Size	N Trial no.	% increases
N2 Wild Type	Control	13.2	0.008	150	3	-
	BAK (6.25µM)	17.11	0.002	150	3	29%
	BAK (12.5 µM)	17.37	0.011	150	3	31%
	BAK (25 µM)	17.44	0.013	150	3	32%
	BAK (37.5 µM)	18.22	0.011	150	3	38%
	BAK (50 µM)	16.94	0.0214	150	3	28%
TK22 (mev-1)	Control	12.56	0.0255	150	3	-
	BAK(37.5 µM)	14.71	0.0366	150	3	17%

No competing interests reported.

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Phenolic monoterpene Bakuchiol ameliorates age related Parkinsonism

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Abstract

Figures

1. Introduction

2. Material and Method

2.1. Maintenance of cultures and chemicals

2.2. Life span assay

2.3. Pharengeal Pumping

2.4. Brood Size

2.5. Intestinal lipofuscin assay

2.6. Juglone survival assay

2.7. Gene Reporter assay of stress response genes SOD-3 & GST-4

2.8. Lipid detection assay by Nile red staining method

2.9. Acridine orange staining assay

2.10. α-Synuclein accumulation level

2.11. 1-Nonanol Repulsion Assay

2.12. Body Bend

2.13. Head Thrash

2.14. Isolation of RNA, synthesis of RNA and cDNA and quantification of mRNA expression

3. Results

3.1. BAK extended the life span

3.2. BAK curtailed the oxidative stress in C. elegans

3.3. BAK demolishes the intracellular ROS level in nematodes

3.4. BAK elevates expression of SOD-3 and GST-4 in C. elegans

3.5. BAK demolishes the lipid content in wild type model of C. elegans

3.6. BAK curtails apoptosis

3.7. BAK attenuates α-Synuclein aggregation level in transgenic strain NL5901

3.8. Enhancement of dopamine level in C.elegans by BAK

3.9. BAK boost locomotary action in C. elegans

4. Discussion

Abbreviations

Declarations

References

Table 1

Additional Declarations

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