With its transparent body, short life span, easy culture methods, ease of maintenance, simple neuronal network, conserved neuronal pathways, and robust genetic screens for investigating molecular mechanisms, C. elegans makes an attractive model in various biological disciplines (Kleawyothatis et al., 2023; Yuan et al., 2023). Table 1 lists the C. elegan strains investigated in our study to assess Nicotine’s therapeutic potential for PD including N2 (Wild-type nematode), OW13 (pkIs2386, unc-54p::human α-synuclein:: YFP + unc-119(+), BZ555) (egIs1, dat-1p::GFP; green fluorescent protein (GFP)), TJ356 (zls356 [daf-16p::da16a/b::GFP + rol-6(su1006)]), CF1553 ([muIs84 ((pAD76) SOD-3p:: GFP + rol-6)]). All C. elegans were purchased through the Caenorhabditis Genetics Centre (CGC), Minnesota, United States. We maintained the worms in solid NGM (nematode growth media) at 20°C and fed them E. coli (OP50) bacteria strains as food. Previously described maintenance procedures were followed for C. elegans (Fu et al., 2014; Muhammad et al., 2022). By treating gravid adults with hypochlorite (0.5 M NaOH and 2% sodium hypochlorite), fertilized eggs were obtained. To obtain the desired larval stage to treat with nicotine in the various experiments given below, synchronized eggs were placed onto OP50-seeded fresh NGM plates or liquid M-9 and incubated at 20°C for 24 h.

Nicotine (Lot#DST180802-032, CAS#54–11-5, HPLC≥98%) was purchased from DESITE (China), 6-hydroxydopamine hydrobromide (6-OHDA; Lot#MKCC1473) was purchased from Sigma-Aldrich (USA), L-Ascorbic acid (Lot. QGNMA-RN, CAS 50–81-7 > 99.0%) purchased from T.C.I. chemicals (Shanghai, China), 2’-Deoxy-5-fluorouridine (FUDR) Lot.#SYEWM-OF, CAS#50–91-9 > 98.0% was purchased from T.C.I chemicals (Shanghai, China), Enzyme-linked immunosorbent assay (ELISA) kit for dopamine (DA) Lot#L190628620 was purchased from Cloud-Clone Corp (China), Juglone (cas#481–39-0, lot#1104A025 (Solar bio), 2,7, dichlorofluorescein diacetate HPLC≥95% cas#2044-85-1, lot#BCBZ7835) was purchased from Sigma life science Germany.

In this assay C. elegans strains OW13 and BZ555 were tested to assess nicotine’s effect on their physiology and determine nicotine’s appropriate dosage concentrations for experiments in the future (Ru-Huei et al., 2014; Chalorak et al., 2021). After overnight growth, E. coli was resuspended in S-medium at an optimum density (O.D.) of 6.6. Nicotine’s desired concentration was achieved by diluting the drug in an E. coli suspension. A 150 μL volume of the final mixture was added per well in the 96-well plate, an approximately synchronized group of 20–30 L1 animals in 10 μL s-medium was added to an E. coli suspension containing a series of nicotine concentrations, which was incubated for six days in 96-well plates at 25°C. In order to prevent solution evaporation, aluminum foil was used to cover the plates. A Microplate Reader M2 SpectraMax (Molecular Devices, Silicon Valley, CA, United States) was used to measure the OD for six days at 600 nm once/day. Before measuring the OD, each plate was placed on a plate shaker for 10 s. A microscopical analysis determined the fraction of live worms in the wells based on size. Each experiment was repeated three times.

BZ555 (GFP-tagged Dopaminergic Neurotoxic model) C. elegans neurons were subjected to neurotoxin (6-OHDA) to stimulate self-neurodegeneration. Briefly, with some minor alterations to the previously described protocol (Nass et al., 2002; Thirugnanam and Santhakumar, 2022). We added 100 mM 6-OHDA and 20 mM ascorbic acid to OP50/S-medium mix both with and without nicotine. BZ555 worms at L-3 (~200 in number) moved into the treated groups, incubated at 22°C for 1 h, and jiggled slightly every 10 min to induce neurodegeneration. After an incubation period of 1 h, BZ555 were washed three times with washing buffer (M9), moved to the OP50/NGM plates with or without nicotine different concentrations, and incubated again for 72 h at 20°C. OP50/NGM/nicotine plates containing 0.06 mg/mL FUDR (5-fluoro-29-deoxyuridine) (Sigma, and St. Louis, MI) because FUDR stops eggs from hatching in C. elegans and thus reduceses its population on plates. Each experiment was repeated three times.

The DA neurons degeneration assay was performed in BZ555 C. elegans treated with or without 6-OHDA/nicotine at various concentrations following a previously described protocol with a few modifications (Hibshman et al., 2021). Following 72 h of incubation at 20°C, strains of BZ555 were washed three times with M9 buffer to remove adhesive bacteria, mounted on glass slides, immobilized with 100 mM sodium azide, enclosed with a coverslip, and monitored under fluorescence microscope. The BX53 microscope (Olympus Corp., Tokyo, Japan) was used to observe immobilized fluorescent images of BZ555 C elegans. Quantifying fluorescence intensity was done using ImageJ software. Each experiment was repeated three times.

We used an Enzyme-linked immunosorbent assay (ELISA assay) kit to evaluate dopamine content in 6-OHDA and 6-OHDA+nicotine’s different concentrations treated C. elegans groups (Bi et al., 2016). In each group, 6-OHDA treated and 6-OHDA/nicotine treated C. elegans BZ555 L3 larvae were washed three times with M9, then weighed. Then, according to the protocol, we added 100 μL, 1X RIPA+Protease inhibitor and used the Dounce homogenizer on ice for complete lysis of the worms. The worms were centrifuged at 4°C for 10 min at 14,000 × g. In accordance with the kit standard protocol, the supernatant was centrifuged and then analyzed by ELISA for DA content. Each experiment was repeated three times.

It is thought that the protein (α -Syn) in OW13 is one of the primary causes of PD. As a result, we chose OW13 (GFP-tagged Muscular α-synuclein expression), a transgenic C. elegans PD model. OW13 worms integrated with human α-syn protein plus GFP fusion construct expressed α-Syn in the muscle’s walls via green fluorescence signals. The method described previously has been modified slightly (Liu et al., 2015; Wang et al., 2020). A synchronized larva stage1 (L-1) strain of C. elegans was transferred onto NGM plates with or without nicotine at 100, 150, and 200 μM and seeded with OP50 and incubated at 20°C for 72 h. 0.06 mg/mL FUDR was added to each NGM drug treated and untreated plates. Nicotine was not administered to control groups. Worms were washed thrice with M9 buffer, mounted on a glass slide, immobilized with sodium azide 100 mM, and enclosed with coverslips after 72 h. Following that, we analyzed α-syn green fluorescent protein (GFP) expression intensity in the treated and untreated worms under the BX53 fluorescent microscope (Olympus Corp., Tokyo, Japan). Fluorescence intensity was quantified using ImageJ software. Each experiment was repeated three times.

For quantitative lipid deposits analysis, in OW13 we used intracellular lipid droplets detective specific fluorescent staining dye Nile red. For stock solution preparation the previously described protocol has been modified slightly (Muhammad et al., 2022). We prepared a 0.5 mg/mL stock solution with 0.5 mg Nile red dye dissolved in 1 mL acetone. As a next step, the stock solution was mixed with E. coli in 1:250, then spread over the NGM plates containing different nicotine concentrations along with 0.06 mg/mL FUDR, and incubated overnight at 37°C. A further 72 h incubation was performed for L-1 C. elegans on NGM/Nile Red/OP50/FUDR plates with or without nicotine at 100,150, and 200 μM. Wild-type N2 C. elegans was used as a negative control. After 72 h, the worms were washed three times with M9, mounted on a glass slide, immobilized with sodium azide 100 mM, and enclosed with coverslips. The fluorescence intensity of lipid deposition was measured in C. elegans using a fluorescence microscope (Olympus, BX53, Japan) and the intensity was quantified using ImageJ software. Each experiment was repeated three times.

There is evidence that dopamine is responsible for C. elegans’ basal slowing response (de Guzman et al., 2022). To evaluate DA neuron integrity, food-sensing behavior assays were performed. When C. elegans are surrounded by food sources, they move slower than when they are without them. We modified the previously described protocol slightly to prepare the NGM plates (Malaiwong et al., 2019). Two types of NGM plates were designed, one with food source (E. coli) and one without (E. coli). Following intoxication with 6-OHDA alone, and 6-OHDA+different nicotine concentrations for 1 h at 20°C, with 10 min intervals of mixing, the BZ555, L3 larva were washed three times with M9. A total of 30–50 C. elegans were placed in 10 μL M9 on NGM plates with and without bacterial lawns. In both bacterial and non-bacterial lawns, locomotory rate of the worms was assessed after 5–10 min of transfer by counting body bending at 20-s intervals. By calculating percentages of locomotion rates in bacteria lawns over non-bacterial lawns, the slowing rate was estimated. We calculated and compared the average slowing rates of 15–20 worms in each group for each analysis. Each experiment was repeated three times.

A previously described protocol with minor modifications was used in order to evaluate the level of free oxidative radicals in-vivo, using 2,7-dichlorofluorescein diacetate (H2DCF-DA, Sigma) (Pandey et al., 2019). A synchronized population of L1 larvae of OW13 was cultured on nicotine-treated and untreated NGM plates for 72 h at 20°C. C elegans were washed three times with M9 after 72 h. 50 C. elegans were shifted to 96-well plates in 150 μL of phosphate buffer solution (PBS,) and 150 μL of 100 mM H2DCF-DA was added per well before reading. Using a microplate reader at 485 nm excitation and 570 nm emission, the fluorescence reading was taken after 2 h. Each experiment was repeated three times.

Based on the protocol previously described, a thermotolerance assay was performed with slight modifications (Yang et al., 2018). On the nicotine-treated and untreated NGM plates, we cultured approximately 50 synchronized OW13, L1 larvae for 7 days at 20°C. Incubation at 37°C for 3 h was started on day 7 of the experiment. Touching each worm with platinum wire every hour was used to score the dead and alive worms. Each experiment was repeated three times.

In the BZ555 strain, we measured nicotine oxidative stress resistance using Juglone (5-hydroxy-1,4-naphthoquinone), an intracellular reactive oxygen species (ROS) generator using the previously mentioned protocol with modifications (Reigada et al., 2020). Synchronized L1 of BZ555 was exposed to 100,150,200 μM of our drug nicotine for 72 h. at 20°C. Control group is BZ555 with without nicotine treatment. After 72 h, the C. elegans were washed three times with M9. 50 μL M9 containing 40–50 C. elegans were poured into 96 wells, and various drug concentrations and 250 μM Juglone were added. Incubated for 24 h. A touch-provoked movement was used to assess the survival percentage of worms after 24 h. Responses to the mechanical stimulus were counted as alive, and vice versa. Each experiment was repeated three times.

A previously described protocol, modified slightly, was used to determine the effect of nicotine on the nuclear localization of DAF-16 in TJ356 zls356 [daf-16p::da16a/b::GFP + rol-6(su1006)] (DAF-16:: GFP fusion protein) transgenic L1 worms (Lee et al., 2018). Synchronized L1 larvae were put on nicotine-treated/untreated NGM plates incubated for 72 h at 20°C. In the following steps, C. elegans were washed three times with M-9 buffer, mounted on a glass slide containing 100 mM sodium azide, and covered with a coverslip. DAF-16::GFP nuclear localization was observed under a fluorescence microscope (Olympus BX53) based on the discrete fluorescence aggregation phenotype. ImageJ software was used for analyzing the images (Carl Zeiss, Gottingen, Germany). Each experiment was repeated three times.

CF1553([muIs84 ((pAD76) SOD-3p:: GFP + rol-6)];) a transgenic strain expressing SOD-3::GFP, was used for the reporter gene assay. To study nicotine’s effect on CF1553 strains, the previously described protocol with modifications was used (Yang et al., 2018). The L-1 synchronized C. elegans were transferred to solid media plates with or without nicotine at 100, 150, and 200 μM and incubated for 72 h at 20°C. Nicotine is not administered to the control group. In the following steps, C. elegans were washed three times with M-9 buffer, mounted on a glass slide containing 100 mM sodium azide, and covered with a coverslip. The fluorescence intensity of the treated group’s images was observed using a fluorescence microscope (Olympus, BX53, Japan). ImageJ was used to calculate the fluorescence intensity of C. elegans images. Each experiment was repeated three times.

We used SPSS/ANOVA (one-way), ImageJ, and GraphPad PRISM, Version 8.02 (1992–2019 GraphPad Software, Inc., La Jolla, CA, United States) for statistical analysis. DA neurons’ GFP intensities were calculated as means ± SD, and their p-values were calculated. Likewise, for α-synuclein, the means ± SD of YFP intensity was also calculated. Each experiment was repeated at least three times and normalized to control groups. Significance differences (p-values) were observed via SPSS/(one-way) ANOVA, followed by Tukey’s or Dunnett’s (where comparison was only to the untreated group) post-hoc tests. In all the experiments, p ≤ 0.001 to p ≤ 0.005 is considered statistically significant and represented by (p ≤ 0.005)*.

We performed a food clearance test to assess nicotine toxic concentrations in C. elegans. We treated transgenic (OW13) and pharmacological (BZ555) worm strains with nicotine at 100, 150, 200, and 250 μM. A concentration of nicotine was tested to determine whether nicotine would be non-toxic to the physiology and hatching capabilities of C. elegans. In our experiment, worms treated with nicotine at 250 μM dilution showed toxic behavior, resulting in smaller bodies and fewer offspring. Even adults die after day intervals. Therefore, it lowers the clearance curve for food. The rest of the treated groups were alive and in good physical health. The addition of nicotine to cultures containing BZ555 and OW13 at 100, 150, and 200 μM concentrations did not affect food clearance compared to 250 μM Figure 2. In our subsequent experiments, we used the highest concentration of 200 μM.

C. elegans possesses eight DA neurons, comprising one pair of anterior deirid (ADE) neurons, two pairs of cephalic (CEP) neurons in the head position, and one pair of posterior deirid (PDE) neurons in the posterior lateral region. C. elegans strain (BZ555) tagged with GFP at the dat-1 promoter is used as a PD model to study the active role of DA neurons. The promoter (Pdat-1: GFP) was used to observe significant changes in DA neurons’ GFP intensity in the head region of the worm. The neurotoxin 6-OHDA was used to generate selective degenerations of DA neurons, which mimicked one of the symptoms of PD in BZ555 C. elegans. To analyze nicotine efficiency, we evaluated neuronal integrity by quantifying the loss of expression of a GFP reporter gene in DA neurons of 6-OHDA-treated BZ555 animals. 6-OHDA treatment reduced GFP expression in PDE neurons and partially lost GFP expression in CEP and ADE neurons Figure 3A. Fluorescence intensity in 6-OHDA-exposed worms decreases by 93% (p ≤ 0.005) compared to non-exposed groups. When nematodes were subjected to nicotine, remarkable protection was observed in DA neurons with CEP, and ADE neurons showed enhanced GFP expression, providing evidence for nicotine’s neuroprotective effects. Furthermore, nicotine at 100, 150, and 200 μM enhanced the fluorescence intensity of GFP in 6-OHDA-intoxicated BZ555 by 14, 33, and 40%, respectively Figure 3B.

C. elegans are devoid of the SNCA orthologous gene. In contrast, nematode genetic flexibility enables the transgenic overexpression of human SNCA genes in muscle walls to investigate SNCA accumulation (Holmqvist et al., 2014). We selected C. elegans OW13 transgenics for examining protein α-Syn aggregation. As a result of the strain’s advantages, OW13 displays higher expression levels and progressive motility defects like PD. Consequently, it demonstrated in vivo aggregative toxicity of protein α-Syn (Muhammad et al., 2022). To observe the active role of nicotine in decreasing α-Syn accumulated toxicity, we treated the worms with or without nicotine. Our assessment verified that nicotine at 100, 150, and 200 μM significantly reduced α-Syn aggregates fluorescence intensity by about 53, 56, and 78% (p ≤ 0.005) compared to untreated groups, dose-dependently. ImageJ software quantifies α-Syn numbers and aggregate volumes of fluorescence intensity Figure 4.

Protein α-Syn has associations with lipid contents and fatty acid modifications, and together they start vesicle formation via a definite mechanism (Jadiya et al., 2011). In OW13 worms, the lipid contents are significantly reduced due to α-Syn existence (Chalorak et al., 2018). α-Syn toxicity disrupted lipid composition in the worms. Moreover, the toxic nature of accumulated α -Syn leads to lipid peroxidation (Saewanee et al., 2021). Lipid molecules are not only responsible for supporting a cell’s structure and constructing membrane components, they are also involved in CNS communication. After nicotine treatment at different concentrations in OW13, we analyzed the lipid content in untreated and nicotine-treated OW13 strains through Nile red staining. In our experiments, nicotine increased the level of lipids by 6.0, 26, and 33% (p ≤ 0.005) dose-dependently Figure 5. A negative control, N2, was also used to compare the lipid content of transgenic OW13 with N2. The lipid content of N2 was 51% higher than that of untreated OW13.

Researchers have demonstrated that animals exposed to 6-OHD exhibit a phenotype of DA neurodegeneration, which subsequently affects dopamine synthesis (Eo et al., 2019). Migration of C. elegans is determined by their bodies bending frequently, which determines their speed (Ezcurra et al., 2011; Kress et al., 2014). On NGM media, C. elegans could sense food sources, usually bacterial lawns. DA neuronal circuits are responsible for C. elegans’ basal slowing rate with food. Consequently, degenerative DA neurons lead to a lower dopamine level, which lowers the ability of nematodes to sense food. Compared with worms treated only with 6-OHDA, the locomotory rate would decrease if nicotine could decrease the 6-OHDA-induced DA neuron degeneration. Nicotine is examined for its ability to save nematodes’ food-sensing behavior under 6-OHDA treatment. As a next step, we examined defects in food-sensing (counted as a “slowing rate”) in 6-OHDA treated worms (BZ555). As compared to control without 6-OHDA treatment, the slowing rate of BZ555 nematodes was decreased 70% (p ≤ 0.005), demonstrating DA neuron dysfunction causes a deficit in food-sensing behavior. We observed that nicotine in BZ555 diminished DA neuron degeneration and significantly increased the slowing rate at (100 μM, by 18%), (150 μM, by 49%), and (200 μM, by 86%) (p ≤ 0.005), dose-dependently. These data collectively proved that nicotine could restore 6-OHDA-mediated DA neuron dysfunction Figure 6A.

We used an ELISA assay to measure dopamine levels in 6-OHDA and 6-OHDA / nicotine-treated BZ555 strains. We observed that the 6-OHDA-intoxicated strain’s dopamine level was reduced compared to the untreated strain. The dopamine levels in 6-OHDA-intoxicated strains reduced by 63% (p ≤ 0.005) compared to untreated control. We observed that nicotine significantly increased dopamine levels by 21, 45, and 58% in 6-OHDA intoxicated BZ555 worms dose-dependently (p ≤ 0.005) Figure 6B.

Per the redox theory of aging, oxidative stress plays a pivotal role in aging (Luceri et al., 2018). A meta-analysis of 47 epidemiologic studies of PD validates the rise of PD incidence with age progression (Collier et al., 2017; Simon et al., 2020). There is also evidence that α-syn aggregation contributes to mitochondrial ROS production (Hsu et al., 2021). An aim of the study was to determine whether nicotine has an antioxidative effect. The ROS labeling dye H2DCF-DA was used to investigate this. Our experimental observation confirmed that nicotine at 100, 150 and 200 μM reduced ROS productions by 34, 63 and 69% dose-dependently as compared to control worms (p ≤ 0.005) Figure 7A.

A previous study has shown that nematode thermal stress is relevant to increasing oxidative stress (Koch et al., 2019). In order to investigate the effect of nicotine on thermotolerance, all treated and untreated worms were moved to 37°C for three hours, and the number of live and dead OW13 worms was counted. The nicotine-treated worms survived longer at 100 μM(13%), 150 μM(22%), and 200 μM(30%) than the untreated worms Figure 7B.

The effectiveness of a drug under oxidative stress is often related to its ability to extend its life span and to act as an antioxidant (Pandey et al., 2019). It has been shown that worms have an increased antioxidant capacity when their antioxidant system is activated (Shi et al., 2023). We tested the antioxidant capability of nicotine by exposing different treated and untreated BZ555 C. elegans to juglone, an oxidative stressor. Juglone/nicotine-treated worms at 100 μM,150 μM, and 200 μM survived longer by 11, 26 and 30% than untreated worms (Juglone only) Figure 7C.

Under stress conditions, DAF-16, a C. elegans homolog of the forkhead transcription factor (FOXO), is activated and different protective genes are expressed (Link et al., 2016; Zecic and Braeckman, 2020). DAF-16 normally resides in the cytoplasm. In order to activate downstream target genes, activated DAF-16 is transferred from the cytoplasm to the nucleus (Mukhopadhyay et al., 2006). The nuclear localization of DAF-16 can be induced by oxidative stress (Senchuk et al., 2018). TJ356 transgenic worms were used to investigate the effect of nicotine on DAF-16 nuclear translocation. Nicotine-treated worms at 100 μM, 150 μM, and 200 μM enhanced DAF-16 translocation from cytoplasm to nucleus by 14, 31, and 49% in a dose-dependent manner (p ≤ 0.005) Figure 8.

SOD activity plays a key role in scavenging ROS. The high antioxidative properties of a drug contribute to its ROS scavenging capacity (Van Raamsdonk and Hekimi, 2012; Niu et al., 2021). In CF1553 worms, sod-3::gfp encodes superoxide dismutases. The effects of nicotine on the CF1553 worms were determined by treating them with nicotine and determining its antioxidative properties. A dose-dependent increase in SOD-3 expression was found by nicotine in CF1553 worms as compared to control (CF1553 without any drugs) at (100 μM,10%), (150 μM, 19%), and (200 μM, 23%), (p ≤ 0.005) Figure 9.