Restoration of MPTP-induced dopamine and tyrosine hydroxylase depletion in the mouse brain through ethanol and nicotine

doi:10.21203/rs.3.rs-4097975/v1

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Restoration of MPTP-induced dopamine and tyrosine hydroxylase depletion in the mouse brain through ethanol and nicotine

https://doi.org/10.21203/rs.3.rs-4097975/v1

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Here, we investigate whether ethanol (EtOH) and nicotine (Nic) alone or in co-exposure can restore the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced depletion of dopamine (DA), DA metabolites, and tyrosine hydroxylase (TH) in the striatum and hippocampus of C57BL/6N mice. MPTP-treated mice were treated intraperitoneally with saline (control), EtOH (1.0–3.0 g/kg), Nic (0.5–2.0 mg/kg), or a combination of EtOH and Nic. Brain samples were collected 1 h after treatment. DA and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA) were measured by HPLC-ECD, while TH expression and Ser31 phosphorylation were quantified by Western blot. EtOH (2.0 and 3.0 g/kg) alone reversed the effects of MPTP treatment in both studied brain regions, as evidenced by an increase in DA, DOPAC, and HVA contents, TH expression, and its phosphorylation at Ser31 compared to the MPTP group, indicating restorative effects on DA neurons in the MPTP model. Likewise, Nic (1.0 and 2.0 mg/kg) alone reversed MPTP treatment effects, with treated mice showing increased DA, DOPAC, and HVA contents, TH expression, and Ser31 phosphorylation compared to MPTP mice. Co-administration of EtOH (2.0 g/kg) and Nic (1.0 mg/kg) further increased DA, DOPAC and HVA tissue contents, TH expression, and Ser31, indicating an additive effect. These results show that moderate to high doses of EtOH and Nic induce similar increases in brain DA and TH via TH phosphorylation activation in MPTP model mice. EtOH and Nic showed an additive effect in combination, suggesting that their co-application could be a potent therapeutic strategy for treating PD.

Ethanol

Nicotine

MPTP

Dopaminergic function

Mouse brain

Parkinson’s disease (PD) is the most prevalent chronic neurodegenerative disorder, resulting from the progressive loss of dopaminergic neurons in the midbrain substantia nigra (German et al., 1992; Vaillancourt and Mitchell, 2020). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin used for generating PD in animal models. MPTP causes damage to the nigrostriatal dopaminergic neurons, inducing PD-like symptoms in rodents (Heikkila et al., 1985; Yokoyama et al., 2011; Kinoshita et al., 2015). Peripherally administered MPTP enters the brain, which converts to the toxic metabolite 1-methyl-4-phenylpyridinium (MPP⁺). MPP⁺ is taken up into dopamine (DA) neurons by DA transporters and inhibits mitochondrial complex I, leading to dopaminergic neuronal damage (Moore et al., 2005; Meredith and Rademacher, 2011). The MPTP mouse model, particularly the C57BL/6 strain, has been widely accepted as the most used model of PD (Heikkila and Sonsalla, 1987; Bhaduri et al., 2018; Mustapha and Mat Taib, 2021).

Ethanol (EtOH) is one of the most widely used chemicals in the world. It primarily acts as a depressant to the central nervous system (CNS) at higher doses but can also act as a stimulant at lower doses (Gulick and Gould, 2007). EtOH affects multiple neurotransmitter systems, including γ-aminobutyric acid, glutamate, endogenous opioids, acetylcholine, and DA (Vengeliene et al., 2008), which can lead to changes in memory, attention, and locomotion (Silvers et al., 2003; Mira et al., 2019; Wang et al., 2020). Among the various neurotransmitter systems involved in the pharmacological effects of EtOH, DA has received considerable attention due to its potential role in the motivational effects of EtOH (McBride et al., 1991; Engel et al., 1992; Brabant et al., 2014). EtOH induces DA synthesis in the animal brain (Siciliano al., 2017), primarily by activating tyrosine hydroxylase (TH) (Tran et al., 2017). Studies have shown that systemic EtOH (0.75–2.25 g/kg) administration increases extracellular DA in the animal brain (Tang et al., 2003; Melendez et al., 2003). Similarly, direct perfusion of a high concentration of EtOH (500 or 860 mM) in the striatum increases extracellular levels of DA in vivo in rats (Yim et al., 1997) and mice (Jamal et al., 2016). Our recent study found that a high concentration of EtOH (3.0 g/kg) could enhance DA metabolism in the mouse brain, as evidenced by an increase in 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) (Jamal et al., 2022). Studies have shown that in humans, chronic low-to-moderate alcohol consumption is associated with a lower risk of PD (Liu et al., 2013; Zhang et al., 2014), while higher alcohol consumption is correlated with an increased risk of PD (Liu et al., 2013). Therefore, determining whether low to moderate or high doses of EtOH in an acute exposure model can restore DA function in the brains of MPTP-treated mice, which serves as an animal model of PD, is of interest.

Nicotine (Nic) is a widely used psychoactive drug often consumed in combination with other substances, such as EtOH. Nic can produce various CNS effects in both humans and animals (Ksir, 1994; Le Foll and Goldberg, 2009). The central effects of Nic are mediated by changes in the release of neurotransmitters, including DA (Toth et al., 1992; Barazangi and Role, 2001). Many studies have shown that Nic stimulates extracellular levels of DA, particularly in the nucleus accumbens (Mifsud et al., 1989; Kleijn et al., 2011), and enhances DA turnover and metabolism in the animal brain (Andersson et al., 1981; Haikala et al., 1986; Brazell et al., 1990). Nic has also been shown to increase TH levels (Carr et al., 1989; Mitchell et al., 1993; Hiremagalur et al., 1993) and DA release (Turner, 2004; Kleijn et al., 2011). A large body of experimental data supports the neuroprotective effects of Nic in PD models (Quik et al., 2006; Cai et al., 2017; Nicholatos et al., 2018). For example, Nic prevents MPTP-induced loss of striatal dopaminergic neurons and attenuates behavioral deficits (Yang et al., 2019; Cai et al., 2017). Recently, Nic was found to improve MPTP-induced motor impairment and neuroapoptosis and enhance TH activity in the substantia nigra and striatal regions of PD mice (Ruan et al., 2023). There is strong and consistent evidence that Nic is associated with a decreased risk of PD (Hernan et al., 2002; Li et al., 2015). However, the relationship between EtOH intake and PD remains controversial. Epidemiological studies have reported that EtOH is inversely associated with PD (Zhang et al., 2014; Jimenez-Jimenez et al., 2019). Therefore, in addition to Nic, we conducted a study on EtOH to determine its association with PD in mice.

EtOH and Nic are the two most commonly abused substances, and many people use them together. Combined exposure to EtOH and Nic has been shown to increase ventral tegmental DA neurons firing both in vitro (Clark and Little, 2004) and in vivo (Tolu et al., 2017) and enhance the release of DA in the nucleus accumbens compared to either drug alone (Tizabi et al., 2002, 2007). This suggests that the effects of EtOH and Nic may be additive when used together. Therefore, this study aimed to investigate whether administrating EtOH and Nic alone or in combination in mice could protect against MPTP-induced DA loss, a toxin known to induce PD in humans. The rationale for considering EtOH and Nic is based on previous findings that suggest mutual reinforcement between the two drugs (Larsson and Engel, 2004) despite their different mechanisms of action and effects. However, no reports have examined the effects of combining EtOH and Nic in animal PD models, and the mechanisms underlying their effects, as well as whether the combination has a higher effect than either drug alone, have yet to be reported. To address this, we used an MPTP-induced PD mouse model to measure DA and its metabolites DOPAC, 3-methoxytyramine (3-MT), and HVA as well as TH and its phosphorylation at Ser31 in the striatum and hippocampus of C57BL/6N mice. The striatum, which receives dense dopaminergic projections, is a crucial region of the dopaminergic system. DA in the hippocampus plays a vital role in hippocampus-dependent learning and memory (Broussard et al., 2016; Bakhtiarzadeh et al., 2023). Therefore, the depletion of hippocampal DA is likely to contribute to a deficit in long-term potentiation in PD models (Zhu et al., 2012). To reverse the deficits in DA release and TH phosphorylation induced by MPTP, we administered EtOH and Nic alone or in combination to the mice via intraperitoneal (IP) injection. DA and its metabolites were simultaneously analyzed using high-performance liquid chromatography coupled to electrochemical detection (HPLC–ECD), and TH and Ser31 phosphorylation were measured by western blotting. We hypothesized that the co-administration of EtOH and Nic would provide better protection in MPTP-treated mice than each compound alone.

Animals

C57BL/6N mice were purchased from Japan SLC Inc. (Hamamatsu, Shizuoka). Mice were housed in groups of 4 and kept at 23 ± 1°C with 12 h of light exposure (06:00–18:00) per day. All experiments were conducted with male mice. Each mouse was 10–12 weeks in age and weighed 23–26 g. All the animal experiments were approved by the Institutional Animal Care and Use Committee of Kagawa University, Japan. All animal experiments should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments and the National Research Council's Guide for the Care and Use of Laboratory Animals. Besides, all procedures performed in the study involving animals were in compliance with the ARRIVE guidelines.

MPTP injection

Mice were randomly assigned to the A. vehicle group (n = 6) or B. MPTP group (MPTP hydrochloride, Sigma-Aldrich, St. Louis, MO, USA). The vehicle group received an equal volume of 0.9% sodium chloride. The mice in the MPTP group received MPTP (20 mg/kg in saline, IP) injections twice a day with a 1 h interval for two consecutive days, resulting in a total dose of 80 mg/kg per mouse. The MPTP dosing regimen was chosen based on a previous report, with a slight modification (Sonsalla and Heikkila, 1986). Mice remained in cages with free access to food and water. The lesions were allowed to stabilize for 7 days before the mice were subjected to an EtOH or Nic injection. All procedures involving MPTP were conducted in strict accordance with published safety and handling guidelines (Jackson-Lewis and Przedborski, 2007).

Open field activity

The open field activity (OFA) was performed on day 1 before the first vehicle and MPTP injection and on days 3, 5, and 8 after the first vehicle and MPTP treatment. The open field task was conducted in a square Plexiglas box (dimensions: 31 cm length, 29 cm width, 30 cm height), enclosed by white paper. The mouse was placed in the center of the testing chamber and allowed to move freely for 10 min for adaptation, followed by a 10-min recording period. The movements were recorded using an overhead video tracking system and stored on a computer. After the experiment, computer-tracking programs were used to analyze the total distance traveled and velocity over time. The apparatus was cleaned with a cotton pad soaked in 10% EtOH after each 10-min session and dried between each test to eliminate odor trails.

Experimental groups

The MPTP-treated mice were divided into eight groups, with six mice in each group (n = 6/group). The groups were as follows: a) saline, b) EtOH at 1.0 g/kg (20% w/v), c) EtOH at 2.0 g/kg, d) EtOH at 3.0 g/kg, e) Nic at 0.5 mg/kg (freebase), f) Nic at 1.0 mg/kg, g) Nic at 2.0 mg/kg, and h) EtOH at 2.0 g/kg + Nic 1.0 mg/kg. All injections were administered via IP on day 8 after the first injection of MPTP. Mice were euthanized 1 h after injection. The doses of Nic and EtOH were adjusted based on the results of previous work (Gubner and Phillips, 2015; Jamal et al., 2022). EtOH was administered at a concentration that produced physiologically relevant blood concentrations, reaching approximately 8 to 40 mM at 60 min in the EtOH 1.0 to 4.0 g/kg groups, respectively (Jamal et al., 2016). EtOH and Nic hydrogen tartrate (Sigma-Aldrich Corp., St. Louis, MO, USA) was dissolved in physiological saline for all experiments.

Brain tissue preparation

Brains were removed and rinsed with ice-cold isotonic saline. One half of the striatum and hippocampus was placed in a 2-ml tube for ex vivo analysis of DA and its metabolites using HPLC-ECD, and the other half was placed in another 2-ml tube for protein analysis. The tubes were stored at − 80°C until use.

Ex vivo HPLC-ECD

The tissue samples were homogenized using a Polytron® homogenizer (Kinematica AG, Lucerne, Switzerland) in 0.2 M perchloric acid (10 µl/mg of tissue), which included 100 µM EDTA-2Na and 1 ng/µl (10 ul) isoproterenol (Tokyo Company Industry Ltd, Japan) as an internal standard (IS). The samples were kept on ice for 30 min and then centrifuged at 15,000 x g at 4°C for 15 min. The supernatants were filtered through 0.45 µm Minisart sterile filters (Sartorius Stedim Biotech GmbH, Germany) and mixed with 1 M Na-acetate to adjust the pH to 3.0. 10 µL of the resulting solution was injected into the HPLC-ECD to determine the levels of DA and its metabolites DOPAC, 3-MT and HVA. To determine the ex vivo concentrations of DA and its metabolites in the brain, we used an HPLC system equipped with an ECD-300 (Eicom, Japan). The main operative conditions for HPLC were as follows: column (EicompaK SC-5ODS; 3.0 mm × 150 mm), oven temperature of 25°C, detector, oxidation potential (+ 750 mV versus Ag/AgCl reference analytical electrode), mobile phase: 83% citrate-acetate buffer (pH 3.5) containing 17% methanol, 190 mg/l sodium octane sulfonate and 5 mg/l EDTA-2Na at a flow rate of 0.23 ml/min. The samples were analyzed for 30 min. The chromatograms were recorded using PowerChrom software version 2.5 (eDAQ Pty Ltd., Densitone East, Australia). Stock standard solutions of 1.0 ng/µl DA and its metabolites were purchased from Eicom (Japan) and stored at 4°C until use.

Western blotting

The other half of the striatum and hippocampus were homogenized using a Polytron® homogenizer in 0.4 ml of RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Added phenylmethylsulfonyl fluoride, sodium orthovanadate, and protease inhibitor cocktail (4 µl each; Santa Cruz Biotechnology, Inc.) to each tube. After centrifugation at 10,000 × g at 4°C for 10 min, the supernatant was used for Western blot analysis. The protein content of the supernatant was determined using the Bradford assay with bovine serum albumin as the standard (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weight markers (Bio-Rad Laboratories, Inc.), and then transferred to polyvinylidene difluoride membranes. The membrane was blocked overnight with 5% skim milk in phosphate-buffered saline at 4°C and then incubated with the following primary antibodies: rabbit anti-TH (1:4000; Cell Signaling Technology, #2792), rabbit anti-phospho-Ser31 (1:1000; Cell Signaling, #3370), and mouse anti-β-actin (1:2000; Wako Pure Chemical Industries, Ltd., Osaka, Japan). The membrane was then incubated with corresponding horseradish peroxidase-linked secondary antibodies. Band intensities were evaluated using an ImageQuant LAS-4000 chemiluminescent imager (GE Healthcare, Tokyo, Japan). The relative protein expressions were normalized to those of β-actin in each sample.

Statistical analysis

Data were expressed as the mean ± SEM. Statistical analyses were performed by using a one-way analysis of variance (ANOVA) followed by a post hoc Tukey–Kramer test. The Student’s t-test was used to compare the MPTP and vehicle groups. The significance level for the post hoc tests was set at P < 0.05. All analyses were conducted using GraphPad Prism software version 5.0 (GraphPad, La Jolla, CA, USA). P values less than 0.05 were considered statistically significant. The percentage change from the vehicle was calculated using the formula: (vehicle – MPTP) / vehicle × 100. The percentage change from MPTP was calculated using the formula: (MPTP − treated) / MPTP × 100.

Effects of MPTP on locomotion, body weight, and mortality

The OFA test evaluated spontaneous motor activity in a novel environment. No notable differences in OFA were found between mice treated with MPTP and those treated with a vehicle (MPTP-free) on days 3, 5, and 8, as shown in Fig. 1A–B. Therefore, the MPTP-treated mice exhibited motor behavior similar to the vehicle group. No motor behavioral measures were performed in mice subjected to similar MPTP and drug treatments. Body weights were measured before MPTP treatment and on day 7 after initiation of MPTP treatment. No considerable difference in body weight was observed between MPTP and vehicle mice on day 7 after MPTP treatment, as shown in Table 1A. These findings are consistent with a previous study that demonstrated that subacute administration of MPTP (30 mg/kg) had no significant effect on mouse weight or motor impairments in the OFA test (Zhang et al., 2017). MPTP-treated mice had a mortality rate of 9.3%, as shown in Table 1B.

Effects of MPTP on striatal DA and its metabolites, TH and Ser31

Ex vivo analyses (Fig. 2A) revealed a significant decrease in the levels of DA (by 82.5%, p < 0.001, t-test) and its metabolites DOPAC (by 70.4%, p < 0.001), 3-MT (by 45.7%, p < 0.001) and HVA (by 68.1%, p < 0.001) in mice treated with MPTP compared to those in the vehicle-treated mice. Western blotting was performed to measure the protein levels of TH and Ser31 in MPTP-treated mice (Fig. 2B). Consistent with the changes in DA and its metabolites, MPTP intoxication considerably reduced TH (by 26.9%, p < 0.001, t-test) and Ser31 (by 54.9%, p < 0.001) expression levels compared to those in the vehicle-treated mice.

Effects of MPTP on hippocampal DA and its metabolites, TH and Ser31

Ex vivo analyses (Fig. 3A) revealed that MPTP treatment resulted in a significant decrease in the levels of DA (by 27.7%, p < 0.001, t-test) and its metabolite HVA (by 42.8%, p < 0.001) compared to those treated with the vehicle, in which a trace to no detection of DOPAC and 3-MT was observed. Western blotting was performed to measure the protein levels of TH and Ser31 (Fig. 3B). The results showed a significant reduction in TH (41.9%, p < 0.001, t-test) levels in MPTP-treated mice compared to those in the vehicle-treated mice. Similarly, the protein levels of Ser31 (53.1%, p < 0.001) significantly decreased in MPTP-treated mice compared to those in the vehicle-treated mice.

EtOH ameliorated the MPTP-induced striatal deficits in DA and its metabolites, TH and Ser31

As shown in Fig. 4A, administration of EtOH at 2.0 and 3.0 g/kg significantly attenuated the MPTP-induced depletion of ex vivo DA [F(3,18) = 10.979, by 139.4%, p = 0.006, EtOH 2.0 g/kg; by 158.7%, p = 0.001, EtOH 3.0 g/kg, one-way ANOVA], DOPAC [F(3,18) = 13.940, by 135.4%, p = 0.015, EtOH 2.0 g/kg; by 167.5%, p < 0.001, EtOH 3.0 g/kg], and HVA [F(3,18) = 22.248, by 115.9%, p = 0.002, EtOH 2.0 g/kg; by 137.6%, p < 0.001, EtOH 3.0 g/kg] compared to those in the MPTP group. EtOH at 1.0 g/kg had no significant effect on DA (p = 0.940), DOPAC (p = 0.740) or HVA (p = 0.523). We next used western blotting to test the change in TH (Fig. 4B) and Ser31 (Fig. 4C) after EtOH administration in MPTP-treated mice. EtOH administration significantly restored the MPTP-induced depletion of TH [F(3,18) = 40.110, by 134.2%, p < 0.001, EtOH 2.0 g/kg; 271.1%, p < 0.001, EtOH 3.0 g/kg, one-way ANOVA] and Ser31 [F(3,18) = 38.405, 27.9%, p < 0.001, EtOH 2.0 g/kg; 50.4%, p < 0.001, EtOH 3.0 g/kg] expression compared to those in the MPTP group. EtOH at 1.0 g/kg had no significant effect on TH (p = 0.994) or Ser31 (p = 0.197) expression.

EtOH ameliorated the MPTP-induced hippocampal deficits in DA and its metabolites, TH and Ser31

As shown in Fig. 5A, EtOH at 2.0 and 3.0 g/kg significantly reduced the MPTP-induced decrease in the levels of ex vivo DA [F(3,18) = 12.389, by 59.3%, p = 0.014, EtOH 2.0 g/kg; by 90.1%, p < 0.001, EtOH 3.0 g/kg, one-way ANOVA] and HVA [F(3,18) = 41.442, by 37.1%, p = 0.036, EtOH 2.0 g/kg; by 107.5%, p < 0.001, EtOH 3.0 g/kg] compared to those in the MPTP group. EtOH at 1.0 g/kg had no significant effect on DA (p = 0.947) or HVA (p = 0.280). We next used western blotting to test the change in TH (Fig. 5B) and Ser31 (Fig. 5C) after EtOH administration in MPTP-treated mice. EtOH administration significantly restored the MPTP-induced impairment in TH [F(3,18) = 11.739, 58.6%, p = 0.002, EtOH 2.0 g/kg; 85.6%, p < 0.001, EtOH 3.0 g/kg, one-way ANOVA] and Ser31 [F(3,18) = 27.669, 63.9%, p < 0.001, EtOH 2.0 g/kg; 91.3%, p < 0.001, EtOH 3.0 g/kg] expression. EtOH at 1.0 g/kg had no significant effect on TH (p = 0.277) or Ser31 (p = 0.692) expression.

Nic ameliorated the MPTP-induced striatal deficits of DA and its metabolites, TH and Ser31

Ex vivo analysis (Fig. 6A) showed that Nic treatment at doses of 1.0 and 2.0 mg/kg significantly attenuated the MPTP-induced decline of DA [F(3,17) = 8.010, by 99.2%, p = 0.046, Nic 1.0 mg/kg; by 145.3%, p = 0.017, Nic 2.0 mg/kg], DOPAC [F(3,17) = 14.469, by 106.6%, p < 0.001, Nic 1.0 mg/kg; by 121.8%, p < 0.001, Nic 2.0 mg/kg], and HVA [F(3,17) = 9.692, by 88.1%, p = 0.044, Nic 1.0 mg/kg; by 133.9%, p = 0.001, Nic 2.0 mg/kg] contents compared to those in the MPTP group. We next used western blotting to test the change in TH (Fig. 6B) and Ser31 (Fig. 6C) protein levels after Nic administration in MPTP-treated mice. Nic administration significantly restored the MPTP-induced suppression of TH [F(3,17) = 17.325, 80.3%, p = 0.002, Nic1.0 mg/kg; 119.9%, p < 0.001, Nic 2.0 mg/kg, one-way ANOVA] and Ser31 [F(3,17) = 12.963, 23.1%, p = 0.388, Nic 1.0 mg/kg; 61.4%, p < 0.001, Nic 2.0 mg/kg] expression compared to those in the MPTP group. Nic at a dose of 0.5 mg/kg did not alter the expression of either TH (p = 0.959) or Ser31 (p = 0.319).

Nic ameliorated the MPTP-induced hippocampal deficits of DA and its metabolites, TH and Ser31

Ex vivo analyses (Fig. 7A) showed that Nic treatment at doses of 1.0 and 2.0 mg/kg significantly reduced the MPTP-induced depletion of DA [F(3,17) = 8.338, by 63.3%, p = 0.048, Nic 1.0 mg/kg; by 98.7%, p = 0.001, Nic 2.0 mg/kg, one-way ANOVA) and HVA [ (3,17) = 8.547, by 31.3%, p = 0.049, Nic 1.0 mg/kg; by 42.4%, p = 0.045, Nic 2.0 mg/kg] tissue contents compared to those in the MPTP group. Neither EtOH nor Nic dose altered the 3-MT content in either studied brain region. We examined the changes in TH (Fig. 7B) and Ser31 (Fig. 7C) protein levels after Nic administration in MPTP-treated mice via western blotting. Nic administration consistently restored the MPTP-induced suppression of TH [F(3,18) = 15.585, 72.8%, p < 0.001, Nic 1.0 mg/kg; 97.2%, p < 0.001, Nic 2.0 mg/kg, one-way ANOVA] and Ser31 [F(3,18) = 15.712, 61.4%, p < 0.001, Nic1.0 mg/kg; 89.9%, p < 0.001, Nic 2.0 mg/kg] expression compared to those in the MPTP group. Nic at a dose of 0.5 mg/kg did not alter the expression of either TH (p = 0.334) or Ser31 (p = 0.832).

EtOH and Nic combination further ameliorated the MPTP-induced deficits of striatal DA and its metabolites, TH and Ser31

Ex vivo analysis (Fig. 8A) showed that the combination of EtOH (2.0 g/kg) and Nic (1.0 mg/kg) produced a significant increase in DA [F(3,16) = 24.664, by 25.6%, p = 0.048 vs EtOH 2.0 g/kg, one-way ANOVA; by 51.1%, p = 0.003 vs Nic 1.0 mg/kg], DOPAC [F(3,16) = 16.644, by 51.2%, p = 0.021 vs EtOH 2.0 g/kg; by 72.2%, p = 0.004 vs Nic 1.0 mg/kg], and HVA [F(3,16) = 33.799, 21.4%, p = 0.049 vs EtOH 2.0 g/kg; by 39.5%, p = 0.002 vs Nic 1.0 mg/kg] tissue contents compared to EtOH (2.0 g/kg) or Nic (1.0 mg/kg) alone. Western blotting was performed to measure the expression levels of TH (Fig. 8B) and Ser31 (Fig. 8C) in the combined EtOH + Nic group. Similarly, EtOH and Nic led to a significant increase in TH expression [F(3,18) = 10.597, by 45.8%, p = 0.048 vs EtOH 2.0 g/kg; by 89.4%, p = 0.004 vs Nic 1.0 mg/kg, one-way ANOVA] and Ser31 phosphorylation [F(3,18) = 74.627, by 93.5%, p < 0.001 vs EtOH 2.0 g/kg; by 101.2%, p < 0.001 vs Nic 1.0 mg/kg].

EtOH and Nic combination further ameliorated the MPTP-induced deficits of hippocampal DA and its metabolites, TH and Ser31

Ex vivo analysis (Fig. 9A) showed that the combination of the two treatments (EtOH, 2.0 g/kg + Nic, 1.0 mg/kg) resulted in a significant increase in DA [F(3,18) = 20.013, by 46.1%, p = 0.006 vs EtOH 2.0 g/kg; by 42.5%, p = 0.002 vs Nic 1.0 mg/kg, one-way ANOVA] and HVA [F(3,18) = 8.480, 54.6%, p = 0.049 vs EtOH 2.0 g/kg; by 61.3%, p = 0.045 vs Nic 1.0 mg/kg] tissue contents compared to EtOH (2.0 g/kg) or Nic (1.0 mg/kg) alone. Western blotting was performed to measure the expression levels of TH (Fig. 9B) and Ser31 (Fig. 9C) in the combined EtOH + Nic group. Similarly, the combination significantly increased the levels of TH [F(3,18) = 116.087, by 92.3%, p < 0.001 vs EtOH 2.0 g/kg; by 76.5%, p < 0.001 vs Nic 1.0 mg/kg, one-way ANOVA] and Ser31 [F(3,18) = 43.100, by 51.7%, p < 0.001 vs EtOH 2.0 g/kg; by 54.8%, p < 0.001 vs Nic 1.0 mg/kg]. Clearly, these findings show that EtOH and Nic modulate the MPTP-induced deficit of dopaminergic function in combination, suggesting further attenuation in these parameters.

We used mice that were administered MPTP as a sub-acute model to investigate whether EtOH and Nic alone or in combination could restore the depletion of dopaminergic function caused by MPTP in the striatum and hippocampus. To accomplish this, we evaluated several measures, such as DA, DOPAC, 3-MT, HVA, TH, and Ser31 phosphorylation, confirming the effects of EtOH and/or Nic in counteracting the dramatic reduction of brain dopaminergic function after MPTP treatment. We found that MPTP decreased DA, DOPAC, 3-MT, and HVA, and the expression of the DA-synthesizing enzyme TH in both the striatum and hippocampus. However, treatment with EtOH (2.0 or 3.0 g/kg) or Nic (1.0 or 2.0 mg/kg) alone noticeably reversed this effect, as evidenced by increased DA and its metabolite (DOPAC and HVA) tissue contents. Western blotting further supported this result, showing that EtOH and Nic alone increased TH expression and Ser31 phosphorylation. The EtOH- or Nic-induced increase in the levels of DOPAC and HVA indicated that part of the DA was further metabolized to DOPAC and HVA. Notably, combined treatment with EtOH (2.0 g/kg) and Nic (1.0 mg/kg) showed a significant increase in DA, DOPAC, and HVA tissue contents accompanied by an increase in TH expression and Ser31 phosphorylation. This increase was greater when EtOH and Nic were administered concurrently than when these drugs were administered separately. Neither EtOH at 1.0 g/kg nor Nic at 0.5 mg/kg altered any of the dopaminergic parameters studied in the MPTP-treated mice, suggesting that these concentrations are insufficient to restore DA neurons. None of these treatments modified the 3-MT level in either brain region examined. Our findings indicate that EtOH and Nic alone preserve dopaminergic function and TH expression that were lost with MPTP treatment. Notably, the combination treatment resulted in an additive increase, suggesting that the co-application of EtOH and Nic has potential as a treatment strategy to restore dopaminergic function in PD.

Mice that received MPTP (cumulative dose; 80 mg/kg of MPTP) exhibited a significant reduction in the levels of DA and its metabolites. In particular, DA levels in the striatum decreased by 82.5% (Fig. 2A) and in the hippocampus by 27.7% (Fig. 3A), which aligns with a previous study of MPTP-treated mice (Itzhak et al., 1999). In addition, MPTP caused a decrease in TH protein expression, with a 26.9% reduction in the striatum (Fig. 2B) and a 41.9% reduction in the hippocampus (Fig. 3B), consistent with previous studies on MPTP-induced PD mice (von Bohlen und Halbach et al., 2005; Alam et al., 2017). In addition to DA depletion, the neurotoxic effect of MPTP on motor activity depends on several factors, including the administration route, the MPTP dose, sex, and strain (Sedelis et al., 2001). Mice treated with MPTP did not show any motor activity deficits on days 3, 5, and 8 after MPTP treatment in the open field box (Fig. 1B). One possible explanation could be that, in these cases, testing was performed when functional recovery might have already occurred. Interestingly, some reports have indicated that MPTP-treated mice continue to exhibit reduced locomotion and rearing activity even weeks later (Arai et al., 1990; Fornai et al., 2005). However, many other studies have shown no changes in locomotion or even increased activity in MPTP-treated mice (Willis et al., 1987; Chia et al., 1996; Itzhak et al., 1999; Zhang et al., 2017), which supports our findings. Therefore, it is reasonable to suggest that a loss of 82.5% DA alone cannot result in PD-like motor deficits in mice. This is supported by a previous study demonstrating that MPTP-induced motor deficits require a loss of DA and a concurrent loss of norepinephrine (Rommelfanger et al., 2007). Furthermore, another study has indicated that serotonin helps regulate motor activity (Mitra et al., 1992).

In this study, EtOH and Nic were administered on day 8 after development of striatal and hippocampal damage that MPTP caused. This regimen was chosen to investigate whether EtOH and Nic could contribute to the reduced PD incidence. We tested three different doses of EtOH (1.0–3.0 g/kg) and Nic (0.5–2.0 mg/kg) alone or in combination with DA and its metabolites and TH as a new entity in MPTP-treated mice. First, we examined the individual effects of EtOH and Nic and found that combining them had advantages, which led us to test EtOH and Nic combined. With the current dosing schedule of EtOH and Nic, there was a potential restoration of dopaminergic function in the striatum and hippocampus, as both drugs improved function after an injury caused by MPTP. The neurotoxic effects of MPTP likely caused this damage.

Previously, we and other researchers showed that EtOH and Nic alone could increase DA and its metabolites, DOPAC and HVA in the animal brain (Haikala et al., 1986; Brazell, et al., 1990; Saeed Dar and Wooles, 1984; Jamal et al., 2022; Yim et al., 1997). There is abundant evidence to demonstrate the rewarding and reinforcing properties of Nic (Xue et al., 2020; Chellian et al., 2021), which are mediated, in part, by its effects on mesolimbic DA neurons (Sun and Laviolette, 2014; Kleijn et al., 2011). EtOH can potentiate some of the rewarding and behavioral effects of Nic in humans and animals (Rose et al., 2004; Meck, 2007). Similarly, Nic can enhance the reinforcing and DA-activating properties of EtOH (Söderpalm et al., 2000). EtOH and Nic modify the activity of dopaminergic neurons in the ventral tegmental area, potentially leading to increased DA release in the nucleus accumbens and affecting the reward system (Morel et al., 2018). The fact that both EtOH and Nic target similar neurotransmitter systems suggests the possibility of synergistic interactions between the two substances (Tizabi et al., 2007; Truitt et al., 2015). Consistent with this hypothesis, the combination of EtOH and Nic can result in heightened reward and neuroadaptation (Gubner and Phillips, 2015; Waeiss et al., 2020). Therefore, the combined application of EtOH and Nic is of particular interest because, despite their increasing use, there is limited information on the effects of combining these substances in PD models. This paper provides evidence that the combination of EtOH and Nic can have better restorative effects by preserving DA function in mice treated with MPTP. The findings reported here highlight three novel observations.

First, MPTP-treated mice that received EtOH (2.0 or 3.0 g/kg) treatment showed increased levels of DA and its metabolites DOPAC and HVA in the striatum and hippocampus compared to those in the MPTP group (Fig. 4,5A). Similarly, EtOH at doses of 2.0 and 3.0 g/kg increased the expression of TH and Ser31 phosphorylation in the brain regions studied in MPTP-treated mice (Fig. 4,5B-C). Notably, increased availability of TH might lead to higher levels of DA in specific brain regions. Thus, an EtOH-induced increase in DA and its metabolites might result from increased expression and phosphorylation of TH. Several reports, including ours, have shown that EtOH significantly increases DA and its metabolites in the brains of mice and Zebrafish (Chatterjee et al., 2014; Saeed Dar and Wooles, 1984; Jamal et al., 2022). Furthermore, EtOH increased TH protein and activity in mammals, including mice (Baizer et al. 1981) and rats (Masserano et al., 1983). Even high concentrations of EtOH (25–200 mM) can enhance TH protein and mRNA expression in the N1E-115 neuronal cell line in vitro (Gayer et al., 1991). Based on these findings, we suggest that the EtOH-induced increase in DA may occur through increased TH activity via an increase in TH protein expression and TH phosphorylation (Tran et al., 2017). This study is the first to report the effects of EtOH in MPTP-treated mice, and these findings provide substantial evidence that EtOH at medium to high doses (2.0 or 3.0 g/kg) has restorative effects on the loss of striatal and hippocampal DA and TH caused by MPTP.

Evidence indicates that prolonged and excessive consumption of EtOH contributes to various neurodegenerative diseases, including PD (Kamal et al., 2020; Peng et al., 2020). Therefore, a potential link between excessive EtOH intake and the development of PD is being hypothesized. Conversely, EtOH at low to moderate concentrations (10–30 mM) may have protective effects in vitro against various toxicants (Belmadani et al., 2004; Ramlochansingh et al., 2011). In some other studies, EtOH (1.0 g/kg) and its first toxic metabolite, acetaldehyde (AcH, 250 mg/kg), increased MPTP toxicity in the brains of mice (Corsini et al., 1985; Zuddas et al., 1989a). This was evident through the depletion of DA and its metabolites DOPAC and HVA and the loss of TH-immunoreactive cells. However, in these experiments, EtOH or AcH was administered 10 min before MPTP (30 mg/kg), and the treatment of EtOH/AcH + MPTP was repeated after 16 h. Their findings suggest that the effects of EtOH on MPTP neurotoxicity might be related to AcH formation. The discrepancies between those results and ours regarding the effects of EtOH could be attributed to the dose and frequency of MPTP administration, and most importantly, the dose and timing of EtOH administration.

The mechanism of the restorative effects of EtOH on MPTP-induced DA depletion in the brain is largely unknown at this time. A previous study showed that AcH at a dose of 250 mg/kg could increase MPP⁺ retention in the striatum of MPTP-treated mice, likely due to slow clearance of MPP⁺ (Zuddas et al., 1989b). This could allow a large amount of MPP⁺ to be stored inside DA neurons, leading to toxic effects. However, EtOH at a dose of 1.0 g/kg does not appear to modify MPP⁺ retention levels in the striatum compared to those with MPTP alone (Zuddas et al., 1989b), suggesting that EtOH at this dose does not considerably affect the clearance of MPP⁺ in the brain. Therefore, in our study, EtOH at 1.0 g/kg did not restore the deficit of DA and its metabolite contents and TH caused by MPTP in the brain (Fig. 4–5). Based on these findings, we hypothesize that a moderate to high dose of EtOH (2.0 or 3.0 g/kg) reduces interference with the clearance of MPP⁺, leading to a decreased MPP⁺ retention level in the brain, which in turn, may attenuate dopaminergic loss. In addition, EtOH (4.0 g/kg) itself attenuates DA clearance, resulting in increased DA levels (Shnitko et al., 2014). Another possible explanation for the restoration of DA function is that EtOH resists MPTP/MPP⁺ neurotoxicity, possibly by activating TH gene expression or phosphorylation. TH phosphorylation at Ser31 or Ser40 modulates DA availability (Salvatore et al., 2001), and an increase in Ser31 TH phosphorylation may increase TH activity in response to TH loss. Therefore, EtOH could restore damaged dopaminergic neurons induced by MPTP to compensate for DA loss in PD. Additionally, α-synuclein, a presynaptic neuronal protein linked to PD, has been shown to regulate the production of DA in cell cultures through its interaction with TH (Perez et al., 2002). EtOH may also protect neurons against synapse damage induced by α-synuclein (Bate and Williams, 2011), further supporting our study on the effects of EtOH on restoring dopaminergic function in PD mice. In contrast, EtOH can decrease dopaminergic neurons relevant to PD (Eriksson et al., 2013; Peng et al., 2020), possibly by inducing cytochrome P450 2E1 (CYP2E1) (Heit et al., 2013). CYP2E1 metabolizes EtOH into AcH, which can enhance MPTP-induced Parkinsonism in mice (Vaglini et al., 2013). This effect was primarily observed with chronic alcohol consumption or exposure to AcH (Vaglini et al., 2013; Zhang et al., 2014). In the present study, we used an acute EtOH exposure model and found that EtOH restored dopaminergic function in the brains of PD mice. However, further studies are required to ascertain the molecular mechanisms by which EtOH protects dopaminergic function in MPTP-treated mice.

Second, we found that Nic at medium to high doses (1.0 or 2.0 mg/kg) exhibited restorative effects against MPTP-induced DA and its metabolite loss in both the striatum and hippocampus. This was evidenced by a significant increase in DA, DOPAC, and HVA levels compared to those in the MPTP group (Fig. 6,7A). As observed in western blot analysis, Nic enhanced TH expression and Ser31 phosphorylation in both brain regions (Fig. 6,7B-C), which is consistent with our DA result. These findings suggest that Nic was able to significantly restore most of the altered MPTP-induced parameters in the mouse brain. Our results align with several previous studies demonstrating that Nic can increase DA and its metabolites, DOPAC and HVA, in the brains of MPTP-treated mice (Janson et al., 1992; Gao et al., 1998). A similar increase in TH has been noted following Nic administration in mice with Parkinsonism induced by MPTP (Parain et al., 2003; Ruan et al., 2023). There are two possible ways to minimize MPTP neurotoxicity: 1) by inhibiting the formation of the active neurotoxin MPP⁺ from MPTP (Heikkila et al., 1984) and 2) by blocking the uptake of MPP⁺ into the DA neurons (Javitch et al., 1985; Sundström et al., 1986). We hypothesize that the restorative effects of Nic observed in our study in the MPTP model mice may be caused by an increased release of DA, which can compete with MPP + for uptake into the striatal and hippocampal DA neurons. Another mechanism that warrants consideration is the role of the nicotinic acetylcholine receptor (nAChR) in minimizing MPTP/MPP⁺–induced damage and restoring DA in MPTP-treated mice. Nic acts on nAChRs located on the dopaminergic nerve terminals to increase DA release in the brain (Besson et al., 2012). MPTP induces astrocyte activation in the mouse brain, which leads to neuronal death (Hu et al., 2011). Interestingly, Nic can inhibit astrocyte activation caused by MPTP/MPP⁺ via its action at α7-nAChR (Liu et al., 2012). This leads to a decrease in the production of pro-inflammatory factors, thus improving dopaminergic loss. The results further support the hypothesis that Nic can effectively ameliorate dopaminergic damage in the brain of MPTP-induced PD mice.

The third important finding of this study is that the combination of EtOH (2.0 g/kg) and Nic (1.0 mg/kg) increased the tissue contents of DA, DOPAC, and HVA and TH expression and Ser31 phosphorylation compared to those with EtOH (2.0 g/kg) or Nic (1.0 mg/kg) alone in the striatum and hippocampus of MPTP-treated mice (Fig. 8,9). Numerous studies have examined the neurobiological effects of EtOH and Nic co-administration (Morel et al., 2019; Cross et al., 2021). However, little is known about the restorative effects of these two agents on MPTP-induced deficits in dopaminergic function. Therefore, we investigated the combined effects of EtOH and Nic on MPTP-induced deficits in dopaminergic function in the mouse brain. Several studies have demonstrated interactions between EtOH and Nic, where Nic may restore some of EtOH's toxic or adverse effects (Prendergast et al., 2000; Penland et al., 2001; Gould and Lommock, 2003; Tizabi et al., 2005). Conversely, EtOH may reduce nicotine-induced seizures (Korkosz et al., 2006a), enhance the effects of Nic in operant behavior (Popke et al., 2000), or increase nicotine-induced place preference (Korkosz et al., 2006b). EtOH and Nic together have also been shown to exhibit antinociceptive analgesic effects (Campbell et al., 2006). Similarly, combined treatment with EtOH and Nic is synergistic in increasing the firing rate of DA-sensitive neurons in the ventral tegmental area of mice, which can potentiate the stimulatory effects of Nic in rats (Schaefer and Michael, 1992; Clark and Little, 2004). For the first time, we demonstrate that EtOH combined with Nic results in a greater increase in DA response in the brain of MPTP-treated mice compared to the effects of EtOH or Nic alone, suggesting an additive effect. Our results are similar to those of a previous study (Tizabi et al., 2002) that combined systemic EtOH with the central administration of Nic into the ventral tegmental area, which resulted in additive or exaggerated DA release in the nucleus accumbens shell. With the combination of EtOH and Nic, the treatment leads to a greater restoration of dopaminergic function in the brain after damage caused by MPTP compared to either drug alone. This finding and previous reports on the synergistic or additive dopaminergic effects of the EtOH and Nic combination support the hypothesis that combining drugs is essential for restoring DA function in mice treated with MPTP.

When administered alone, moderate to high doses of EtOH and Nic caused a significant increase in DA, DOPAC, and HVA levels and TH expression and Ser31 phosphorylation in the striatum and hippocampus of MPTP-treated mice. When EtOH and Nic were administered together, the drugs further increased DA and its metabolite contents, TH expression, and Ser31 phosphorylation. This suggests an additive restorative effect on the dopaminergic function. These findings provide the first evidence that EtOH and Nic could be promising substances for PD treatment, especially when combined.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contribution

Mostofa Jamal: Writing – original draft, Methodology, and Data curation Sella Takei: Visualization and Investigation Ikuko Tsukamoto: ValidationTakanori Miki: Data analysis Ken-Ichi Ohta: Data curationMd Zakir Hossain: VisualizationHiroshi Kinoshita: Review and Supervision

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research [Grant No. (c) 19K10687] from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Table 1 is available in the Supplementary Files section.

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Table1.tif
Table 1: Effects of MPTP on body weight and mortality. (A) body weight (g), (B) mortality (%).
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Restoration of MPTP-induced dopamine and tyrosine hydroxylase depletion in the mouse brain through ethanol and nicotine

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