Docosahexaenoic acid protects motor function and increases dopamine synthesis in a rat model of Parkinson’s disease via mechanisms associated with increased protein kinase activity in the striatum

Abstract

Parkinson’s disease (PD) is a devastating neurodegenerative disease that leads to motor deficits and selective destruction of nigrostriatal dopaminergic neurons. PD is typically treated by dopamine replacement agents; however, dopamine replacement loses effectiveness in the later stages of the disease. Here, we describe the neuroprotective effects of the omega-3 fatty acid docosahexaenoic acid (DHA) in the medial forebrain bundle 6-hydroxydopamine (6-OHDA) model of advanced-stage PD in rats. We show that daily administration of DHA protects against core symptoms of PD, including deficits in postural stability, gait integrity, and dopamine neurochemistry in motor areas of the striatum. Our results also demonstrate that DHA increases striatal dopamine synthesis via phosphorylation of the rate-limiting catecholamine synthesizing enzyme tyrosine hydroxylase, in a manner dependent on the second messenger-linked protein kinases PKA and PKC. We also show that DHA specifically reverses dopamine loss in the nigrostriatal pathway, with no effect in the mesolimbic or mesocortical pathways. This suggests that DHA is unlikely to produce pharmacotherapeutic or adverse effects that depend on dopamine pathways other than the nigrostriatal pathway. To our knowledge, previous reports have not examined the effects of DHA in such an advanced-stage model, documented that the dopamine synthesizing effects of DHA in vivo are mediated through the activation of protein kinases and regulation of TH activity, or demonstrated specificity to the nigrostriatal pathway. These novel findings corroborate the beneficial effects of omega-3 fatty acids seen in PD patients and suggest that DHA provides a novel means of protecting patients for dopamine neurodegeneration.

1. Introduction

Parkinson’s Disease (PD) is a neurodegenerative disease that is characterized by the selective destruction of nigrostriatal dopamine neurons in the midbrain and loss of dopamine nerve terminals in the striatum, including within the caudate putamen. The symptoms of PD include impairments in motor function, such as rigidity, tremor, and postural instability. While most cases of PD are idiopathic, factors like environmental exposures and genetic susceptibility can induce events tied to the development of PD, such as neuroinflammation and oxidative stress (Bogaerts et al., 2008; Eckert et al., 2013). Indeed, a growing body of evidence suggests that nutrition and diet, including dietary fatty acids, may play an important role in PD (Bousquet et al., 2011a; Mischley et al., 2017; Seidl et al., 2014).

Polyunsaturated fatty acids (PUFA) are essential fatty acids that are known to be components of neuronal cell membranes in the brain (Laye et al., 2018). PUFA appear to have neuroprotective roles and are essential factors in brain growth and development (Simopoulos, 1991). Recent studies have established a link between intake of dietary PUFA and a decreased incidence of neurodegenerative diseases, such as Alzheimer’s disease (Wu et al., 2015). Our current research focuses on the ω3-PUFA docosahexaenoic acid (DHA), an essential ω3-fatty acid that is linked to a myriad of brain functions (Lauritzen et al., 2016). Several potential mechanisms have been suggested for the neuroprotective actions of DHA in a variety of brain injury models, including reduced neural apoptosis and increased synaptogenesis (Block and Hong, 2005), down-regulation of COX-2 expression (Lee et al., 2009), modulation of microglial activity (Chang et al., 2015), and reduced oxidative stress (Huun et al., 2018). PD has a pathophysiological basis that includes progressive nigrostriatal dopamine cell loss, formation of protein aggregates, and increased levels of oxidative stress. Previous studies using PC12 cells have indicated that DHA attenuates oxidative stress induced by H₂O₂ (Che et al., 2018), which could mediate its neuroprotective roles. Studies conducted using endothelial cells revealed that PUFA attenuated oxidative stress by upregulating NRF-2 mediated antioxidant response, which decreased DNA damage (Sakai et al., 2017). Moreover, DHA is known to modulate brain-derived neurotropic factor expression via the extracellular receptor kinase (ERK) and p38-mitogen-activated protein kinase (MAPK) pathways in the context of inflammatory and oxidative diseases (Sona et al., 2018). Despite these findings, there is a limited understanding of the mechanisms through which DHA may act to protect against PD and virtually no understanding of the signaling pathways involved in the anti-PD effects of DHA.

Previous studies have shown that DHA treatment in mouse models of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Bousquet et al., 2009; Ozkan et al., 2016) and rat models of PD induced by intrastriatal 6-OHDA (Cansev et al., 2008) restore dopamine levels and protect motor function. However, the mechanisms of this effect, particularly on tyrosine hydroxylase (TH), the rate-limiting enzymatic regulator of catecholamine synthesis, remain elusive. While control of TH activity over the timescale of days can be achieved through de novo TH protein synthesis, causing an increased capacity for catecholamine synthesis, the acute activity of TH can also be regulated on the order of minutes, via phosphorylation of the N-terminus, an effect that is known to be modulated by the second-messenger kinases including Ca⁺²-dependent protein kinase (PKC) and cAMP-dependent protein kinase (PKA). The N-terminus of TH contains 4 serine residues (Ser⁸, Ser¹⁹, Ser³¹, and Ser⁴⁰) that are capable of being differentially phosphorylated by protein kinases, both in vivo and in vitro, to regulate catalytic activity (Kumer and Vrana, 1996). Chief amongst these, the sustained phosphorylation of TH at Ser⁴⁰ increases TH activity, which in turn directly leads to increased catecholamine synthesis (Dunkley et al., 2004). Given that animal models of PD make use of chemical toxins such as MPTP or 6-OHDA, which lead to striatal degeneration, the goal of the current study was to examine the effects of DHA on TH, and PD symptomology, using an advanced-stage PD model.

2. Materials and methods

2.1. Animals

The test subjects for the PD model were male Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) that weighed between 200 and 300 g and ranged in age from 2 to 3 months at the time of surgery (N = 30). Due to known effects of aging on TH phosphorylation sensitivity, ex vivo striatal mince assays used male Sprague Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) that weighed 125–150 g and were about 1 month old at the time of assay (N = 9). All rats had ad libitum access to food and water and were housed singly on a 12-h light/dark cycle (7:00 AM–7:00 PM) in a temperature regulated room at typical ambient temperatures. The rats were fed Lab Diet 5001 Rodent Diet which contains the following dietary fats composition as described in Table 1 below. All behavioral experiments were conducted during the light phase. All experiments were conducted using protocols that were approved by the Mercer University Institutional Animal Care and Use Committee.

Table 1.

Dietary Fat composition of Lab Diet 5001 Rodent Diet. Data from manufacturer’s online catalog.

Nutrient	Percentage
Fat (ether extract), %	4.5
Fat (acid hydrolysis), %	5.5
Cholesterol, ppm	200
Linoleic Acid, %	1.16
Linolenic Acid, %	0.07
Arachidonic Acid, %	< 0.01
Omega-3 Fatty Acids, %	0.26
Total Saturated Fatty Acids %	1.50
Total Monounsaturated Fatty Acids, %	1.58

2.2. Drugs and chemicals

Hydrochloric acid (HCl, 867790) was purchased from Carolina Biological Supply Company (Burlington, NC). Perchloric acid (HCLO₄, 3752) was purchased from GFS Chemicals (Powell, OH). 3,4-dihy-droxyphenylacetic acid (DOPAC) (850217), dopamine hydrochloride (H8502), desipramine hydrochloride (D3900), 6-OHDA hydrochloride (H4381), Homovanillic acid (HVA) (H1252), bovine serum albumin (BSA) (A2153), L-ascorbic acid (A5960), phorbol 12-myristate 13-acetate (PMA) (16561-29-8), bisindolylmaleimide II (BIM II) (B3056), forskolin (FSK) (F6886) and H-89 dihydrochloride (B1427) were purchased from Sigma Aldrich (St. Louis, MO). Ketoprofen (0215515405) was purchased from MP Biomedicals (Santa Ana, CA). Purified DHA oil was purchased from Nu-Chek Prep (Elysian, MN). All other chemicals used were obtained at the highest available purity from Thermo Fisher Scientific (Waltham, MA) or Sigma-Aldrich (St. Louis, MO). All injections were administered at a volume of 0.001 ml (vehicle) per gram body weight of each rat.

2.3. Experimental design

An illustration of the treatment timeline used in this study is provided in Fig. 1. Rats were randomly assigned to the following four experimental groups: Group I: Negative control group that received a sham surgery and daily vehicle administrations, Group II: Negative control group that received sham surgery and daily DHA administrations. Group III: Positive control group that received an infusion of 6-OHDA into the medial forebrain bundle (MFB) and daily vehicle administrations, Group IV: Treatment group that received an infusion of 6-OHDA into MFB and daily administrations of DHA. The MFB conveys efferent fibers from nigral cell bodies to the striatum and lesioning with 6-OHDA in the MFB causes massive anterograde degeneration of the nigrostriatal pathway (Blandini and Armentero, 2012), which models the later stages of PD. The study timeline comprised a total of 15 days. DHA was administered by intraperitoneal injection (IP) in Group II and Group IV 1 h prior to vehicle o 6-OHDA infusion respectively, and then once a day for 14 consecutive days. Behavioral assessments at the end of the treatment period included automated open-field testing (OFT) (Day 13), the stepping test (Day 14), and stride length analysis (Day 14). On the 15th day, all animals were euthanized, and their brains were extracted for neurochemical and biochemical studies.

Fig. 1.

Timeline of the experimental treatment regimen used in this study

2.4. 6-OHDA and DHA treatment

All animals except the sham surgery control groups were unilaterally lesioned with the selective dopaminergic neurotoxin 6-OHDA to induce parkinsonian symptoms and dopamine depletion (Torres et al., 2011). The 6-OHDA solution was prepared fresh prior to each infusion by dissolving 6-OHDA (6 mg/ml) in sterile saline that contained ascorbic acid (0.02%) as a stabilizer (Thiele et al., 2012), followed by filtering through a 0.22 μm filter. The sham surgery control groups were infused with the vehicle. DHA (300 mg/kg) was suspended in a mixture of bovine serum albumin (BSA) and saline (10 mg BSA per ml saline) and was prepared immediately before each injection, which occurred 1 h prior to the vehicle or 6-OHDA infusion, and then daily for two weeks. The 300 mg/kg dose of DHA was chosen to mimic concentrations seen upon dietary intake (2000 mg daily for humans) and was adjusted for a 10-fold higher metabolic rate for rats as used previously (Gao et al., 2016; Kunder et al., 2017), with BSA included as a carrier (Bonilla et al., 2010; Chang et al., 2015; Diaz et al., 2002; Wiesenfeld et al., 2001). The intraperitoneal (IP) route of administration was selected because it provides tight control over the dose administered in an initial proof of concept study, and because of potential issues associated with oral delivery such as slower delivery onset, interference with absorption based on diet and food consumed, and variability in systemic absorption from the digestive tract (Turner et al., 2011). The two week study period was chosen as it is known that the dopamine depletions and motor symptoms of 6-OHDA lesioning emerge within two weeks of exposure (Jeon et al., 1995; Zuch et al., 2000).

2.5. 6-OHDA lesion surgery

Rats were pretreated with desipramine (12.5 mg/kg, IP) 30 min before 6-OHDA infusion to protect noradrenaline neurons (Chang et al., 2003). Ketoprofen (2 mg/kg, SC) was administered as an analgesic and rats were anesthetized via inhaled isoflurane (1–3% induction, maintenance to effect), which was verified via ablation of pedal withdrawal following toe pinch. The animal was placed on a heating pad throughout the surgical procedure and its body temperature was monitored using a rectal probe. Stereotaxic surgeries were conducted using a Kopf model 963SD motorizer ultra precise (0.5 μm resolution) small animal stereotaxic workstation (David Kopf Instruments; Tujunga, CA) controlled using software by Neurostar (Tubingen, Germany), including the Smart Bregma Finder. The hair on the skull was removed with a hair clipper and the area of surgical incision was disinfected with isopropyl alcohol and povidone-iodine prior to making a midline incision of < 2 cm. A single unilateral infusion of 6-OHDA (5 μl containing 30 μg) (Jang et al., 2012) was carried out in the MFB via a 10 μl micro syringe at the rate of 1 μl per minute into the coordinates AP: −4.0 mm, ML: −1.5 mm and DV: 8.5 mm (Paxinos and Watson, 2013), as shown previously (Jadavji et al., 2006). After every 1 min of infusion, there was a 0.5-min pause to ensure enough diffusion time for the solution. Sham-operated rats received the same volume of saline containing 0.02% ascorbic acid using the same procedure. After the completion of the infusion, 5 min of diffusion time was allowed before gentle retraction of the cannula. The incision was then closed with simple interrupted absorbable sutures (5–0 Vicryl), and the area was cleansed with isopropyl alcohol. The animal was kept on a warming pad and observed until awakening from anesthesia.

2.6. Behavioral studies

2.6.1. Automated open-field test (OFT)

Automated analysis of locomotor activity in the OFT was performed using Activity Monitor 5 by Med Associates Inc (St Albans, VT) in a standard Med Associates rat OFT chamber (44 × 44 × 30 cm) mounted with infrared detectors, which was housed in a light and sound attenuating cubicle. The chamber was of sufficient size to allow each animal to move freely while being tracked. After the animal was placed in the center of the testing arena, locomotor activity was quantified as the number of beam interruptions (as detected by crossings), and was recorded by a computer as total distance travelled and ambulatory counts over a period of 10 min (Yu et al., 2018). After each animal was tested, the open-field chamber was cleaned thoroughly with decon solution (0.78%) to avoid any possible interference from previous animal residues or odors.

2.6.2. Stepping test/adjusting steps test

The adjusting steps test has been extensively used to measure postural stability in rats (Kane et al., 2011; Olsson et al., 1995; Woodlee et al., 2008). Unilateral lesioning with 6-OHDA produces limb asymmetry in lesioned rats as indexed by deficits in forelimb stepping on the side contralateral to the lesion in comparison to the ipsilateral side. The adjusting step count was determined with slight modifications to procedures previously described (Olsson et al., 1995). The rat was handled by the experimenter for at least 3 days prior to actual testing to acclimate the rat to the procedure. The rat was held by its torso while one of its forelimbs and both of its hind limbs were fixed above the surface of the table. This position led to the body weight of the rat being entirely supported by just the other forelimb in contact with the surface. The rat was then moved backwards along a 50 cm platform at a steady pace, which caused the free forelimb in contact with the surface to make adjusting steps in order to maintain its balance on the platform. The number of self-initiated steps made with each paw was video recorded and analyzed offline by a trained and blinded experimenter. A total of three trials were conducted and an average of the adjusting steps initiated across the three trials was calculated for each paw and compared.

2.6.3. Gait analysis

Gait disturbance is one of the primary symptoms seen in PD patients (Moustafa et al., 2016). Stride length has been used to assess abnormal gait in rats (Su et al., 2018). For this procedure, the rat’s hindlimb paws were dipped in non-toxic paint and the rat was placed on a runway measuring 10 × 7 × 15 cm with high side walls to confine the rat within the runway, with slight modifications to procedures previously described (Metz et al., 2005). The floor of the runway was lined with ordinary white paper. As the rat freely walked through the runway, the floor was marked with its footprints. The distance between the hind paws for the ipsilateral and contralateral stride were recorded and expressed as the ipsilateral and contralateral stride length (cm) respectively. Two sets of stride length values were collected from the central portion of the runway and expressed as average values.

2.7. Neurochemical measurements

Fifteen days post-lesioning, rats were anesthetized by CO₂, decapitated and their brains rapidly removed and rinsed in cold phosphate buffered saline; with all subsequent steps performed at 4 °C. Brains were sliced into 1 mm thick coronal sections using a rat brain matrix and were placed flat on a cold metal plate over ice. Striatal punches were taken from the lesioned and unlesioned hemisphere from individual slices using a 2 mm diameter tissue biopsy punch, as we have previously described (Murnane et al., 2012; Ray et al., 2018). The specific tissue processing procedures were customized and described separately for each individual neurochemical and biochemical assay in the following sections. Samples underwent neurochemical analysis as previously described (Murnane et al., 2012; Ray et al., 2018), using ultra high-performance liquid chromatography (HPLC) coupled to electrochemical detection (ECD). Tissue from the striatum (lesioned and unlesioned striatum) were punched and sonically disrupted in 200 μl of 0.3 N HClO₄ and centrifuged for 10 min at 17,000 g at 4 °C to remove cellular debris. The clear supernatant was filtered through a0.22-μm filter into autosampler vials. The WPS-3000TBSL autosampler was maintained at 10 °C and 10 μl was injected onto a Thermo Scientific Hypersil BDS C18 column (35 °C) with Thermo Scientific Dionex Test Phase running at a flow rate of 0.5 mL/min. Coulometric detection was accomplished with a Thermo Scientific Dionex 6011RS electrode cell. The signal was analyzed on a Thermo Scientific Dionex Chromeleon CDS processing platform. Absolute tissue concentrations (ng/mg) for dopamine and its primary metabolites DOPAC and HVA were determined by comparison with external standard curves and corrected for tissue weight. Dopamine turnover was assessed by calculating the ratio of DOPAC/dopamine and HVA/dopamine.

2.8. Immunoblotting

Immunoblotting was performed as we have described previously (Singh and Moniri, 2012). Briefly, tissue punches from lesioned striatum were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM Na₂HPO_4, pH 7.4) and a protease inhibitor cocktail for 30 min at 4 °C and centrifuged for 10 min at 4 °C at 17,000 g. The supernatant was collected and stored at −80 °C until analysis. On the day of analysis, the protein concentrations were standardized using DC Protein Assay (Bio-Rad, Hercules, CA) and the lysate denatured in 2× SDS sample-buffer and boiled for 5 min. Equivalent concentrations of lysates were resolved by SDS-PAGE followed by a transfer to PVDF membranes. The blots were incubated with appropriate primary antibodies at room temperature for 1-h (except Phospho-TH Ser⁴⁰ and Phospho-TH Ser³¹ which were incubated overnight at 4 °C, per the manufacturer’s recommendation). Blots were visualized with a horse-radish-peroxidase-conjugated secondary antibody followed by Enhanced Chemiluminescence (Fonsart et al., 2008). The following antibodies were used: TH (Sigma Aldrich, AB152), Phospho-TH Ser⁴⁰ (36–8600, Life Technologies), Phospho-TH Ser³¹ (Invitrogen, 36–9900), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SC-25778, Santa Cruz Biotechnology). All primary antibodies were used at 1:1000 dilution in 5% BSA blocking buffer.

2.9. Minces assay for quantification of dopamine

Homogenates of prefrontal cortex, striatum and hippocampal minces were prepared in phosphate buffered saline as described above and treated alone or in combinations of 6-OHDA (100 μM), the PKC activator PMA (1 μM), the PKC inhibitor BIM II (10 μM), the adenylyl cyclase (and hence, downstream PKA) activator FSK (10 μM), the PKA inhibitor H-89 (10 μM), or DHA (100 μM). The minces were pretreated alone or in combinations with PMA, BIM II, FSK and H-89 for 10 min at 37 °C (Burns et al., 2014), subsequent to DHA and 6-OHDA addition for an additional 20 min at 37 °C. The prefrontal cortex and hippocampal minces were just treated with either DHA or 6-OHDA, alone or in combination for 20 min at 37 °C. The tubes were then centrifuged at 17,000 g for 4 min and the supernatant aspirated. The tissue pellet was then lysed in 0.3N perchloric acid and samples were prepared as described in section 2.7.

2.10. Data analysis

All graphical data presentations were created using GraphPad Prism (GraphPad Software Inc.; La Jolla, CA) and results are presented as the mean ± the standard error of the mean (SEM). Statistical analysis was performed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc analysis for samples displaying homogeneity of variance via the Brown Forsythe test. The Kruskal-Wallis test followed by Dunn’s multiple comparisons test was performed as a non-parametric test for samples displaying non-homogenous variance. The minces assay for quantification of dopamine concentrations was analyzed using ANOVA and Bonferroni’s post-hoc test as there were specific preplanned comparisons. The success of the 6-OHDA lesions was verified by performing Grubb’s test for outliers using GraphPad. The test was performed on the dopamine levels in the lesioned striatum of the 6-OHDA animals. One animal was excluded from the study as it was a significant outlier (p < 0.05).Statistical significance is represented as a single symbol for p < 0.05, a double symbol for p < 0.01, and a triple symbol for p < 0.001, as noted in the figure legends.

3. Results

3.1. In vivo assessments in 6-OHDA-lesioned animals

Motor dysfunction is a major hallmark of PD. To assess the effects of DHA on motor function, we tested locomotor activity, limb asymmetry and stride length. Using OFT to assess locomotor activity, our results show no significant effect of 6-OHDA or DHA on total distance travelled or on ambulatory counts (N = 7–8 per group; data not shown). Next, we determined the effects on limb asymmetry, which is known to be dysfunctional in unilateral models of PD (Blume et al., 2009). One-way ANOVA revealed a significant main effect of treatment on limb asymmetry (Kruskal-Wallis statistic = 26.71, p < 0.001). Post-hoc analysis demonstrated that, as expected, 6-OHDA lesioning significantly decreased contralateral paw steps compared to sham vehicle infusions (Fig. 2A). Importantly, 6-OHDA-lesioned animals treated with DHA had significantly improved contralateral steps that mirrored vehicle-sham animals, demonstrating a blockage of the neurotoxin-induced limb asymmetry (Fig. 2A). To further examine the effects of DHA on PD-induced motor deficits, we assessed stride length as a measure of gait. Results showed that 6-OHDA lesioning significantly reduced stride length on both ipsilateral and contralateral sides (F_7,42 = 26.27, p < 0.001), respective to each vehicle sham control (Fig. 2B), and DHA significantly blocked the shortening of stride length in lesioned but untreated animals. Moreover, DHA exhibited significantly improved stride length on both sides, respective to lesioned but untreated animals. Together, these data demonstrate that DHA prevents the development of the motor symptoms of 6-OHDA-induced neurotoxicity (Fig. 2B). To determine if these results are a consequence of DHA-induced elevations in striatal dopamine levels, we directly assessed striatal dopamine levels as well as dopamine turnover. These results shown in Fig. 2C indicate that 6-OHDA significantly reduced dopamine levels in the lesioned striatum, and this effect was significantly prevented with DHA treatment (Kruskal-Wallis statistic = 19.02, p < 0.001). To establish whether this increase in dopamine was related to inhibition of dopamine metabolism, we examined the turnover of dopamine to its metabolite DOPAC and major metabolite HVA. While the results in Fig. 2E show no significant effect of 6-OHDA or DHA on dopamine turnover estimated with the DOPAC/DA ratio (F_3,26 = 1.741), the results in Fig. 2G show a significant increase of dopamine turnover to its major metabolite HVA in 6-OHDA-lesioned rats, which was prevented with DHA treatment (Kruskal-Wallis statistic = 17.17, p < 0.001). This indicates that DHA treatment was effective at inhibiting dopamine metabolism. 6-OHDA lesioning also produced significant depletions in the levels of dopamine metabolites in the lesioned striatum. Fig. 2D indicates a significant effect of 6-OHDA lesioning on DOPAC levels (Kruskal-Wallis statistic = 17.29, p < 0.001). Fig. 2F indicates a significant depletion of HVA levels in the lesioned striatum due to 6-OHDA lesioning (Kruskal-Wallis statistic = 12.39, p < 0.01).

Fig. 2.

A) Effects of DHA on limb asymmetry as evaluated by the stepping test. 6-OHDA treatment significantly (p < 0.001) decreases contralateral paw steps relative to vehicle group. 6-OHDA-le-sioned animals treated with DHA exhibit significant improvement in contralateral paw steps (p < 0.01). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, as assessed by one-way ANOVA and post-hoc Dunn’s multiple comparison test (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). B) Effects of DHA on gait as evaluated by bilateral stride length. 6-OHDA lesioning significantly shortens both ipsilateral and contralateral stride length (p < 0.001) versus vehicle lesioning. 6-OHDA-lesioned animals treated with DHA exhibit significant improvement in stride length (p < 0.001) versus 6-OHDA-lesioned but untreated animals.* = p < 0.05, ** = p < 0.01, *** = p < 0.001, for bilateral stride length compared to vehicle, # = p < 0.05, ## = p < 0.01, ### = p < 0.001, for bilateral stride length compared to 6-OHDA group as assessed by one-way ANOVA and post-hoc Tukey’s multiple comparison test (N = 6 for Vehicle group, N = 4 for Vehicle + DHA group, N = 7 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). C) Effects of DHA on striatal dopamine levels in lesioned striatum. 6-OHDA significantly (p < 0.001) depletes dopamine levels in the lesioned striatum which significantly (p < 0.05) improves in animals treated with DHA treatment. Data shows that* = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to vehicle group, # = p < 0.05, compared to 6-OHDA group as assessed by one-way ANOVA and post-hoc Dunn’s multiple comparison test. One rat from the 6-OHDA group was removed from the analyses of the study as it was found to be an outlier by the Grubb’s test thereby indicating partial lesioning and was hence excluded from the study (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). D) Effects of DHA on DOPAC levels in lesioned striatum. 6-OHDA significantly (p < 0.05) depletes DOPAC levels in lesioned striatum which significantly (p < 0.01) improves with DHA treatment. Data shows that * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to vehicle group, # = p < 0.05, ## = p < 0.01 compared to 6-OHDA group as assessed by one-way ANOVA and post-hoc Dunn’s multiple comparison test (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 9 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). E) Effects of DHA on DOPAC/dopamine turnover in lesioned striatum. No significant difference found across groups as assessed by one-way ANOVA (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). F) Effects of DHA on HVA levels in lesioned striatum. 6-OHDA significantly (p < 0.01) depletes HVA levels in lesioned striatum. Data shows that * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to vehicle group, as assessed by one-way ANOVA and post-hoc Dunn’s multiple comparison test (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). G) Effects of DHA on HVA/dopamine turnover in lesioned striatum. 6-OHDA significantly (p < 0.05) increases HVA/dopamine turnover in the lesioned striatum which significantly (p < 0.05) decreases in animals treated with DHA treatment. Data shows that * = p < 0.05 compared to vehicle group, # = p < 0.05, compared to 6-OHDA group as assessed by one-way ANOVA and post-hoc Dunn’s multiple comparison test (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). All DHA injections were administered intraperitoneally at a dose of 300 mg/kg and at a volume of 0.001 ml drug solution (dissolved in vehicle) per gram body weight of each rat.

The development of a successful unilateral lesioning model was verified by evaluating levels of dopamine and its metabolites in the unlesioned striatum (Fig. 3). There were no significant differences found in the dopamine levels in the unlesioned striatum across the groups (F_3,26 = 2.339) (Fig. 3A). Similarly, there were no significant differences in dopamine turnover to its metabolites DOPAC (DOPAC/dopamine turnover; F_3,26 = 0.442) (Fig. 3C) and HVA (HVA/dopamine turnover; F_3,26 = 0.9584) (Fig. 3E) in the unlesioned striatum across the groups. Additionally, there were no significant differences in DOPAC levels (F_3,26 = 1.203) (Fig. 3B) or HVA levels (F_3,26 = 0.1701) (Fig. 3D) across the groups in the unlesioned striatum.

Fig. 3.

A) Effects of DHA on dopamine levels in unlesioned striatum. No significant difference found across groups as assessed by one-way ANOVA (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). B) Effects of DHA on DOPAC levels in unlesioned striatum. No significant difference found across groups as assessed by one-way ANOVA (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). C) Effects of DHA on DOPAC/dopamine turnover in unlesioned striatum. No significant difference found across groups as assessed by one-way ANOVA (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). D) Effects of DHA on HVA levels in unlesioned striatum. No significant difference found across groups as assessed by one-way ANOVA (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). E) Effects of DHA on HVA/dopamine turnover in unlesioned striatum. No significant difference found across groups as assessed by one-way ANOVA (N = 8 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 10 for 6-OHDA group, N = 8 for 6-OHDA+DHA group). All DHA injections were administered intraperitoneally at a dose of 300 mg/kg and at a volume of 0.001 ml drug solution (dissolved in vehicle) per gram body weight of each rat.

Given that DHA-induced protection of dopamine neurochemistry and motor function was associated with inhibition of dopamine metabolism, we probed the effects of DHA on de novo synthesis of dopamine by investigating the effects of 6-OHDA lesioning and DHA treatment on the expression and phosphorylation of TH in the lesioned striatum. Results in Fig. 4C demonstrate that 6-OHDA lesioning significantly decreases (p < 0.001) the total expression of TH, which is rate-limiting towards dopamine synthesis. Importantly, 6-OHDA-lesioned animals treated with DHA had near normal levels of TH expression, demonstrating that DHA is neuroprotective of dopamine neurons that express the biosynthetic enzyme (F_{3, 53} = 20.17; p < 0.0001) (Fig. 4C). Similarly, DHA-treated animals had non depleted levels of phospho-Ser⁴⁰-TH (Fig. 4B), which is the active enzyme involved in dopamine synthesis and correlates precisely with our data in Fig. 2, suggesting that both increased dopamine synthesis and decreased metabolism may contribute to preserved motor function (Kruskal-Wallis statistic = 23.24, p < 0.0001 main effect of treatment on phospho-Ser⁴⁰-TH levels) (Full-length blot images shown in supplementary material Figure figs1, figs2, figs3, figs4, figs5 and figs6). Importantly, there were no significant differences across the three groups for phosphor-Ser³¹ expression (data not shown), suggesting that the phosphorylation of Ser⁴⁰, which is directly catalytically-relevant is specifically modulated by DHA activity.

Fig. 4.

A) Representative blot showing effects of DHA on TH expression and activity. A representative immunoblot is shown, and data from at least 3 independent experiments was quantified using images obtained from Image Lab software and then quantified using Image J software to give band intensities. Expression of TH and TH-Ser⁴⁰ was normalized to the housekeeping gene GAPDH and is expressed as the mean ± SEM of the independent experiments. B) 6-OHDA treatment effectively abolishes striatal phospho-TH-Ser⁴⁰ expression, which represents active TH (p < 0.001 versus vehicle). Lesioned animals treated with DHA had significantly higher total expression of TH phospho-Ser⁴⁰-TH (p < 0.05 versus 6-OHDA). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to vehicle group, # = p < 0.05, when compared to 6-OHDA group as assessed by one-way ANOVA and post-hoc Dunn’s multiple comparison test (N = 6 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 4 for 6-OHDA group, N = 4 for 6-OHDA+DHA group). C) 6-OHDA treatment effectively abolishes striatal total TH expression in lesioned animals (p < 0.001 versus vehicle). Lesioned animals treated with DHA had significantly higher total expression of TH (p < 0.001 versus 6-OHDA). * = p < 0.05, ** = p < 0.01, *** = p < 0.001 compared to vehicle group, ### = p < 0.001, when compared to 6-OHDA group as assessed by one-way ANOVA and post-hoc Tukey’s multiple comparison test (N = 6 for Vehicle group, N = 4 for Vehicle+ DHA group, N = 4 for 6-OHDA group, N = 4 for 6-OHDA+DHA group) (for both graphs; Fig. 4B and C). All DHA injections were administered intraperitoneally at a dose of 300 mg/kg and at a volume of 0.001 ml drug solution (dissolved in vehicle) per gram body weight of each rat.

3.2. Ex vivo assessments

Given our findings using a 6-OHDA model demonstrate that DHA increases dopamine synthesis and protects motor function, we investigated mechanisms underlying these effects using striatal minces. To begin to probe potential signaling pathways underlying the beneficial effects of DHA, we examined the roles of kinases that are known to modulate striatal TH activity and dopamine synthesis. Interestingly, our results show that the facilitation of dopamine synthesis by DHA is ablated upon treatment of striata with BIMII (Fig. 5A), an inhibitor of PKC, as well as H-89, an inhibitor of PKA (Fig. 5B) (F_{6, 42} = 13.99; p < 0.0001), demonstrating a role for both kinases in the mechanism of DHA-modulated dopamine synthesis. Moreover, PKA activation recapitulated the dopamine restorative effects of DHA, while PKC activation did not, suggesting that DHA-mediated dopamine synthesis is directly stimulated through the former. These results corroborate with the known modulation of TH-Ser⁴⁰ phosphorylation directly by PKA, while PKC is known to only indirectly effect this residue, via downstream PKA activation (Haycock and Haycock, 1991; Lindgren et al., 2000). Together, these studies suggest that the anti-Parkinsonian effects of DHA seemingly involve the activation, and crosstalk of PKC to PKA. We then decided to evaluate if the DHA induced dopamine synthesis was specific to the striatal minces or occurred in other brain regions such as prefrontal cortex and hippocampus. Interestingly, our results demonstrate significant depletions in dopamine levels in both prefrontal cortex minces (Fig. 5C) (F_3,8 = 17.74; p < 0.001), as well as hippocampal minces (Fig. 5D) (F_3,8 = 8.264; p < 0.01). Furthermore, DHA treatment did not restore dopamine levels in these minces, suggesting DHA stimulates distinct mechanisms of dopamine synthesis in the striatum as compared to the prefrontal cortex and hippocampus which should be investigated further.

Fig. 5.

A) Effects of PKC on DHA-induced dopamine synthesis in striatal minces. For dopamine level in striatal minces, one-way ANOVA revealed a significant main effect on dopamine level across the various groups (F_6,42 = 13.99, p < 0.0001). Post-hoc analysis by Bonferroni’s multiple comparisons revealed that 6-OHDA treatment significantly (p < 0.001) reduced dopamine level in striatal minces which significantly (p < 0.001) improved with DHA treatment. In contrast, pre-treatment with BIM II (p < 0.001) significantly lowered the dopamine level. ** = p < 0.01, *** = p < 0.001. All results were expressed as percent control of the saline group. B) Effects of PKA on DHA-induced dopamine synthesis in striatal minces. For dopamine level in striatal minces, one-way ANOVA revealed a significant main effect on dopamine level across the various groups (F_6,42 = 13.99, p < 0.0001). Post-hoc analysis by Bonferroni’s multiple comparisons revealed that 6-OHDA treatment significantly (p < 0.001) reduced dopamine level in striatal minces which significantly improved with FSK pre-treatment (p < 0.05) and DHA treatment (p < 0.001). In contrast, pre-treatment with H-89 (p < 0.01) significantly lowered the dopamine level. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. All results were expressed as percent control of the saline group. Results represent pooled data from at least three independent experiments with similar control values (Fig. 5A and B: N = 6, represents the number of striatal minces used from 6 individual rats). C) Effects of 6-OHDA and DHA in prefrontal cortex minces. For dopamine level in prefrontal cortex minces, one-way ANOVA revealed a significant main effect on dopamine level across the various groups (F_3,8 = 17.74; p < 0.001). Post-hoc analysis by Bonferroni’s multiple comparisons revealed that 6-OHDA treatment significantly (p < 0.01) reduced dopamine level in prefrontal cortex minces which did not improve with DHA treatment. ** = p < 0.01. All results were expressed as percent control of the saline group. Results represent pooled data from at least three independent experiments with similar control values (N = 3, represents the number of prefrontal cortex minces used from 3 individual rats). D) Effects of 6-OHDA and DHA in hippocampal minces. For dopamine level in hippocampal minces, one-way ANOVA revealed a significant main effect on dopamine level across the various groups (F_3,8 = 8.264; p < 0.01). Post-hoc analysis by Bonferroni’s multiple comparisons revealed that 6-OHDA treatment significantly (p < 0.05) reduced dopamine level in hippocampal minces which did not improve with DHA treatment. * = p < 0.05. All results were expressed as percent control of the saline group. Results represent pooled data from at least three independent experiments with similar control values (N = 3, represents the number of hippocampal minces used from 3 individual rats).

4. Discussion

Emerging literature suggests a neuroprotective role of DHA in animal models of PD (Bousquet et al., 2011b; Cansev et al., 2008; Hernando et al., 2019; Tanriover et al., 2010), yet few studies have examined the mechanisms underlying these neuroprotective effects of DHA. For example, although DHA has been shown to restore dopamine neurotransmission (Cansev et al., 2008; Shin and Dixon, 2011), the mechanisms through which DHA increases dopamine synthesis remain undescribed. TH is the rate-limiting enzyme in dopamine biosynthesis and is regulated by multiple kinases (Daubner et al., 2011), including PKA and PKC. It is unclear whether DHA directly modulates TH expression or regulates TH activity indirectly through kinases. In the present study, we report that DHA increases dopamine levels and protects motor function in a MFB 6-OHDA model of advanced-stage PD. DHA appears to enhance dopamine synthesis in the striatum, along with affecting turnover of dopamine to its major metabolite HVA and increasing total TH expression and phosphorylation of TH-Ser⁴⁰ in striatal tissue. Since BIM II and H-89 inhibited the ability of DHA to protect dopamine, a major and novel finding in our study is that PKC and PKA activity appears to be necessary for the beneficial effects of DHA. Since DHA did not restore dopamine levels in prefrontal cortex minces or hippocampal minces, the present data also suggest the effects of DHA are specific to the striatum. Together, these findings suggest that the anti-parkinsonian effects of DHA may be linked with striatal PKC and PKA kinase activity in vivo. Studies have reported that PKA and PKC signaling mechanisms are implicated in several processes, including regulation of TH activity (Piech-Dumas et al., 2001) and attenuating the generation of ROS (Savitha and Salimath, 1993).This suggests that DHA is utilizing signaling pathways in the striatum specifically that seemingly involve kinase activity. Interestingly, recent studies have reported that ω3-fatty acids like DHA are ligands for cell-surface freefatty acid receptors (Moniri, 2016), suggesting that DHA may modulate these receptors to trigger the activity of PKA or PKC and produce downstream protection of dopamine neurochemistry and motor function.

Loss of striatal dopamine is a hallmark symptom of PD (Galvan and Wichmann, 2008) and is believed to mediate the impairments in motor function that are present in patients with this disease (Boix et al., 2018). To model the later stages of PD, a relatively large dose of 6-OHDA was targeted to the MFB. Additionally, it was hypothesized that DHA may play a neuroprotective role by preventing the neurochemical depletions and motor deficits induced by 6-OHDA infusions. Animals treated with 6-OHDA, but not DHA, displayed > 99% depletion of dopamine levels, as well as frank motor deficits in postural stability and gait coordination compared to controls. However, both the loss of dopamine neurochemistry and the motor deficits induced by OHDA were prevented with DHA treatment—a result which suggests DHA may be a promising treatment for advanced cases of PD.

Although DHA significantly prevented stride length shortening and prevented limb asymmetry, neither 6-OHDA nor DHA altered gross locomotor activity in the OFT. Previous reports suggest that gross locomotor impairments following 6-OHDA lesioning are more evident after longer time periods (Silva et al., 2016; Su et al., 2018). Thus, a deficit in gross locomotor activity would have possibly emerged if more time was allowed between the surgery and the assessment.

We hypothesized that the stride length would exhibit asymmetry post unilateral lesioning (Hsieh et al., 2011). However, we observed significant decreases across both stride lengths (Boix et al., 2018; Glajch et al., 2012; Iancu et al., 2005) probably due to the lesioned rats adapting their ipsilateral stride length to the shorter contralateral stride length, thereby displaying bilateral shortened stride length as an adaptative mechanism.

The present data support the notion that DHA has protective effects on motor function (Gomez-Soler et al., 2018; Perez-Pardo et al., 2017). It has also been reported that DHA supplementation in a mouse PD model does not affect motor symptoms like bradykinesia, despite increases in the number of TH positive cells (Ozsoy et al., 2011). Thus, an increase in the number of TH cells is not sufficient to improve motor deficits and DHA may be acting through distinct, nonspecific mechanisms. Considering this possibility, the effects of DHA on dopamine neurochemistry were evaluated. We report that DHA—at a concentration and treatment regimen that had no significant effect on dopamine levels in sham rats or in the contralateral non-lesioned hemi-sphere—protects dopamine neurochemistry in lesioned rats. We also report that DHA appears to increase dopamine synthesis along with slowing dopamine metabolism. An increase in dopamine striatal turnover is a classic component of striatal dopamine depletions in the 6-OHDA-lesioned rat model (Zigmond et al., 1989) and has been reported in the early stages of PD (Brotchie and Fitzer-Attas, 2009; Kurlan et al., 1991; Sossi et al., 2004). While we did not observe an increased turnover of dopamine into its metabolite DOPAC, we observed an increased dopamine turnover into its major metabolite HVA, which we further observed was prevented with DHA treatment. Several studies have reported that the HVA/dopamine ratio is generally greater than that of DOPAC/dopamine as HVA is the major metabolite of dopamine (Altar et al., 1992; Pifl et al., 2014). As our model represents late stage PD, and over 99% loss of striatal dopamine, along with significant depletions in dopamine metabolites (Agrawal et al., 2012; Goes et al., 2014; Rizelio et al., 2010; Walker et al., 2013) it is possible that we would observe increased turnover of dopamine to DOPAC with other induction paradigms or with post-surgical assessment periods shorter than two weeks.

Short term regulation of dopamine biosynthesis occurs via modulating the phosphorylation of TH, which is involved in the conversion of tyrosine to DOPA (Haycock, 1993). TH Phosphorylation at Ser⁴⁰ is the major site for the modulation of its activity (Bobrovskaya et al., 2007; Dunkley et al., 2004). In our study, DHA treatment significantly increased both the expression of TH and phospho-TH (Ser⁴⁰), suggesting that DHA may be directly or indirectly increasing TH activity by stimulating the phosphorylation of TH (Ser⁴⁰) through other receptors and kinases, and subsequently increasing dopamine biosynthesis. Moreover, we did not find any significant differences in the expression of phospho-TH (Ser³¹) across the groups in our study, and hence, we conclude that DHA predominantly leads to the phosphorylation of TH only at the catalytically relevant site Ser⁴⁰. The capacity of DHAto be involved in the phosphorylation of the major regulatory site of TH, the rate limiting enzyme involved in dopamine biosynthesis supports DHAas a viable treatment for PD.

Given that TH activity is regulated by multiple kinases, we used striatal minces to assess whether dopamine synthesizing effects of DHA could be mediated via PKA or PKC signaling pathways. TH can be phosphorylated at Ser⁴⁰ in situ directly by PKA in response to FSK or directly or indirectly by PKC in response to phorbol esters like PMA (Dunkley et al., 2004). In this experiment, DHA lost its capacity to restore dopamine levels when it was pretreated with BIM II, a PKC inhibitor. Importantly, the PKC activator PMA did not recapitulate the dopamine restorative effects of DHA, demonstrating that PKC activation is necessary but not enough for induction of dopamine synthesis. Similarly, DHA could not restore dopamine levels in the presence of PKA inhibitor H-89. Interestingly, the downstream PKA activator FSK recapitulated the dopamine restorative effects of DHA to some extent, validating that direct stimulation of TH at Ser⁴⁰ by PKA increases enzyme activity leading to dopamine restorative effects, as established previously (Daubner et al., 2011; Dunkley et al., 2004; Sura et al., 2004; Tekin et al., 2014). While the exact nature of the cross talk between the PKA and PKC pathways are not clearly understood, it has been reported (Piech-Dumas et al., 2001) that treatment of PC12 cells with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, a PKC activator, stimulates TH gene promoter via signaling pathways that are dependent on PKA. Our data suggests that there could be similar crosstalk between the PKA and PKC pathways following activation with DHA, which may lead to the pronounced dopamine protective effects of DHA. Further studies are underway in our laboratories to examine this hypothesis. The treatment of prefrontal cortex minces and hippocampal minces with 6-OHDA produced significant depletion of dopamine (Hauber et al., 1994; Moreno-Castilla et al., 2017). Interestingly, DHA treatment did not restore dopamine levels in the prefrontal cortex minces or the hippocampal minces, suggesting that DHA acts differently in the striatum to synthesize dopamine. Similar results were reported where fish oil supplementation did not change dopamine levels in the cortex or hippocampus of adult Wistar rats (Vines et al., 2012). This correlates with our data suggesting that DHA may be acting in the striatum by increasing TH activity by stimulating the phosphorylation of TH (Ser⁴⁰), which seemingly requires the activation of both PKA and PKC pathways and increases dopamine biosynthesis in the striatum.

Additionally, it is possible that DHA blocked the toxic effects of 6-OHDA by preventing its neuronal uptake or by altering its metabolism, which would produce less oxidative stress and/or greater clearance; however, this was not examined in the present study. Since 6-OHDA lesioning causes significant oxidative stress and ROS generation (Puspita et al., 2017), via monoamine oxidase-mediated H₂O₂ formation in vivo (Eisenhofer et al., 2004; Meiser et al., 2013), it is likely that DHA could be decreasing 6-OHDA mediated neurotoxicity by reducing ROS levels. Perhaps these anti-oxidant effects of DHA against ROS formation are mediated by signaling pathways involving PKA/PKC, as has been shown previously in leukocytes (Savitha and Salimath, 1993) and in macrophages (Cheshmehkani et al., 2017).

Another point worth consideration is the dose and route of DHA administration used in the current study. While the 300 mg/kg dose of DHA was selected based on recommended human doses (Gao et al., 2016), orally administered DHA would likely require a higher dose to provide similar biological drug exposure to the dose administered through an IP route of administration. An IP route of administration was selected for the current proof of concept study to allow tighter control on DHA dosing as discussed in detail in Section 2.4. However long-term studies in rodents and translational clinical studies in humans will require administration of DHA orally, and oral administration should be considered in future studies now that the concept that DHA can reverse the deficits seen in a robust PD model has been established.

Fatty acids like arachidonic acid have been reported to interact with dopamine transporters and inhibit the transport of dopamine (Chen et al., 2003; Keith et al., 2011). One study documented that co-treatment of l-DOPA with oral DHA at 100 mg/kg for a month lowered parkinsonian scores in MPTP monkeys as compared to control subjects and prevented the increase of preproenkephalin and preprodynorphin mRNA in the striatum (Tamim et al., 2010), which is observed in untreated MPTP monkeys. However, DHA treatment had no significant effects on improving depleted dopamine transporter binding in the caudate nucleus and putamen of MPTP lesioned monkeys. In a study using MPTP mice, it was reported that exposing mice to a high omega-3 (n-3) PUFA diet prior to MPTP exposure prevented the MPTP-induced depletion of Nurr1 mRNA and dopamine transporter mRNA levels in the substantia nigra (Bousquet et al., 2008). Yet, there was no significant effect of the high omega-3 (n-3) PUFA diet on transporter specific binding in the striatum of the MPTP mice. Despite the absence of a significant effect on the transporter specific binding in the striatum, the study reported protective effects of the diet on striatal dopamine levels, thereby supporting neuroprotective roles of (n-3) PUFA in PD models. Another study, this time using female Wistar rats, reported that (n-3) PUFA dietary deficiency affects dopamine transporter density and function in the frontal cortex, but not the striatum (Kodas et al., 2002). Taken together, these results suggest that (n-3) PUFA may have region specific neuroprotective effects on dopamine transporters in PD models. Furthermore, metabolites of DHA such as N-docosahex-aenoylehanolamide (DEA), resolvins and neuroprotectin D1 (NPD1) could be involved in some neuroprotective actions of DHA as reported previously (Bazan et al., 2011; Kim et al., 2011). The role of TH in PD has been an area of great interest (Haavik and Toska, 1998; Tabrez et al., 2012). Several studies have reported decreased levels of TH and TH phosphorylation dysregulations in the SN of aged rats (Salvatore et al., 2009) due to factors like neuroinflammation (Capuron et al., 2011), oxidative stress (De La Cruz et al., 1996) and α-synuclein accumulation (Zhu et al., 2012). While our ex vivo experiments on TH phosphorylation utilized young rat striatal minces, the in vivo experiments display significantly improved levels of Total TH and TH Ser⁴⁰ with DHA treatment in adult 6-OHDA-lesioned rats. Thus, it appears likely that DHA would influence TH phosphorylation in older rats, and further studies are needed to ascertain this. Nonetheless, our results show a clear dopamine-synthesizing and neuroprotective role for this fat in the 6-OHDA model.

In summary, we report that DHA protects dopamine levels and motor function in the 6-OHDA model. DHA treatment significantly increased total TH expression as well as phosphorylation on TH-Ser⁴⁰. In striatal tissue, protection of dopamine was linked to activation of both PKC and PKA. The antiparkinsonian effects of DHA appear to be linked to induced PKC and PKA kinase activity and this study is one of the first to implicate in vivo signaling pathways involving PKA and PKC in the anti-parkinsonian effects of DHA as well as to implicate differential effects of DHA in the striatum.

Thus, this study builds on the literature by elucidating signaling pathways related to induce dopamine synthesis, specifically in the striatum. Future studies should continue to study the mechanisms involved in the regulation of TH by DHA, as a deeper understanding of the effects of DHA on these molecular processes could help in clarifying the role of DHA in the dopamine nigrostriatal pathway, could illuminate the physiological processes regulating dopamine synthesis, and could aid in the potential development of DHA and perhaps other PUFA as therapeutics for PD.

HIGHLIGHTS.

• DHA protects motor function in advanced-stage rat model of PD

• DHA increases dopamine synthesis in advanced-stage rat model of PD

• Dopamine synthesizing effects of DHA are mediated through TH and protein kinases

• Dopamine synthesizing effects of DHA demonstrate specificity to nigrostriatal pathway

Abbreviations:

PD

Parkinson’s disease

PUFA

polyunsaturated fatty acids

DHA

docosahexaenoic acid

TH

tyrosine hydroxylase

PKC

Protein Kinase C

PKA

Protein Kinase A

6-OHDA

6-hydroxydopamine

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MFB

medial forebrain bundle

DOPAC

3,4-dihydrox-yphenylacetic acid

HVA

Homovanillic acid

BSA

bovine serum albumin

PMA

phorbol 12-myristate 13-acetate

BIM II

bisindolylmaleimide II

FSK

forskolin

OFT

open-field testing

HPLC

high-performance liquid chromatography

ECD

electrochemical detection

ROS

reactive oxygen species