Valeric Acid Protects Dopaminergic Neurons by Suppressing Oxidative Stress, Neuroinflammation and Modulating Autophagy Pathways

The dosage of Val used in this study did not show any toxicity and it was selected based on previous study [40]. Antioxidant enzymes plays a major role in scavenging reactive oxygen species which is a product of oxidative metabolism. Four weeks after rotenone administration, we found an robust decrease in vital antioxidants (Catalase, Glutathione and Superoxide dismutase) and significant increase in malondialdehyde (MDA) levels, an marker of lipid peroxidation. However, oral administration of Val after rotenone exposure significantly increased antioxidant levels with concomitant decrease in MDA (Figure 1). These results demonstrate that Val can scavenge rotenone-induced oxidative stress in rats.

We next examined the ability of Val to modulate the production and secretion of inflammatory factors in rotenone induced PD model. It is well established that pro-inflammatory cytokines augment neurodegenerative mechanisms. Our results showed that rotenone injection induced a significant increase in production of pro-inflammatory factors which is evident from increase in interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), nitric oxide (NO) and matrix metalloproteinase 9 (MMP-9) (Figure 2A,B). Notably, administration of Val potentially diminished the production of pro-inflammatory factors thereby establishing Valeric acid mediated anti-inflammatory mechanism. In addition, enhanced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been reported in the brain of PD patients and also causes dopaminergic neuronal loss in PD models [11,41]. Consistent with previous reports, rotenone administration caused a significant increase in expression of iNOS and COX-2, whereas Val treatment markedly reduced these protein levels as evinced by western blotting (Figure 2C,D).

Enhanced expression of glial markers, especially ionized calcium-binding adapter molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP), is an indicator of reactive microgliosis and reactive astrocytes respectively. These two cells play a major role in inflammation mediated neurodegeneration in PD. Figure 3 shows that immunofluorescent staining for Iba-1 and GFAP activated microglia and astrocytes respectively in the striatum of experimental animals. Activation of microglia by rotenone is evident from larger cell bodies and fewer processes (Figure 3A). Similarly, astrocyte activation by rotenone is denoted by enhanced expression of GFAP positive cells (Figure 3C). Interestingly, administration of Val in rotenone treated rats significantly reduced the expression of Iba-1 and GFAP in the striatum of experimental animals. These results were in concordant with the diminished production of pro-inflammatory factor production described previously. Quantification of activated microglia and astrocyte are represented as percentage of control and depicted as histogram in Figure 3B,D respectively.

Loss of tyrosine hydrolase (TH) positive neurons in the substantia nigra pars compacta (SNpc) and the resultant decrease of TH expression in the striatum are hallmark pathological events in PD. Hence, we measured TH+ve dopaminergic neurons in SNpc and its expression in the striatum. Administration of rotenone for 4 weeks, caused a significant reduction in the number of TH positive neurons (Figure 4A,B) which in turn caused 50% decrease in the intensity of TH+ve striatal fibers (Figure 4C,D). Significantly, we found that administration of Val to rotenone toxicated rats prevented dopaminergic neuronal loss and enhanced TH expression in striatal fibers as demonstrated by immunohistochemistry.

Accumulation of α-synuclein in Lewy bodies is an key pathological event in PD and enhanced expression of α-synuclein influences neuronal death by either necrosis or apoptosis [42]. Moreover, impairment of autophagy results in accumulation of aggregated α-synuclein protein favoring neurodegeneration. We found that rotenone administration caused a significant increase (3 fold) in α-synuclein protein levels in the substantia nigra pars compacta as shown by western blotting (Figure 5). However, we noted that treatment with Val caused a significant decrease in α-synuclein expression. These results indicate Val partly protects dopaminergic neurons by diminishing the expression of α-synuclein in rotenone treated animals.

Various reports have shown that impairment of mTOR activity leads to neuronal dysfunction and have adverse effect on neuronal regenerative mechanisms [43,44]. p70 S6 kinase (p70S6K) is one of the most defined downstream effector molecules of mTOR. In central nervous system, aggregated protein toxicity initially increases mTOR activity but its expression is subsequently reduced leading to cell death [45]. In this study, we wanted to evaluate if Val treatment could prevent neuronal apoptosis by reinstating mTOR activity. Immunoblotting analysis showed that rotenone administration caused a significant decrease in mTOR, phospho mTOR and p70S6K expression (Figure 6A,B). Whereas, Val administration restored mTOR activity by enhancing the expression of these proteins. In addition, to evaluate the importance of mTOR activity on neuronal survival and to assess if Val could regulate apoptosis signaling pathway, we analysed the levels of Bax and Bcl-2 by western blotting. We found that rotenone administration caused a significant increase in the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2. By contrast, Val administration prevented apoptosis by diminishing the expression of Bax and enhancing Bcl-2 expression (Figure 6C,D). Together, these results suggest that Val prevented rotenone mediated apoptosis by reinstating mTOR pathway.

As mentioned earlier, accumulation of misfolded α-synuclein due to impairment of autophagy plays a central role in lewy body formation and resultant neurodegeneration in PD. Hence, to further delineate neuroprotection mechanisms of Val, we analyzed whether Val could modulate rotenone impaired autophagy using MAP-light chain 3 (LC3) and p62 protein expression levels. Treatment with rotenone caused a significant increase in autophagosome accumulation which is demonstrated by enhanced LC3II/LC3I ratio. Whereas, Val administration to rotenone intoxicated rats significantly reduced LC3II/LC3I ratio demonstrating decrease in autophagosome accumulation. (Figure 7A,B). To further assess the role of Val in autophagy regulation, we assessed the expression of p62 which helps to link ubiquitinated proteins to autophagic process through LC3. Administration of rotenone caused a significant increase in p62 levels demonstrating that rotenone inhibits autophagic degradation. Alternatively, treatment with Val caused a significant decrease in p62 expression levels suggesting that it favors autophagic degradation of misfolded proteins (Figure 7C,D). In addition, the ratio of LC3II/LC3I and p62 levels did not significantly increase in both control and Val alone treated groups.

All experimental animal procedures were approved by Animal Ethics Committee of United Arab Emirates University (UAEU). Adult male Wistar rats (6–7 months old) weighing 280–300 g were used in this study. Animals were maintained at pathogen free facility with ambient conditions (22 ± 1 °C, 60% humidity, and 12 h diurnal cycle) and had ad libitum access to food and water. Animals were allowed 1 week initially to adapt to the experimental room conditions.

Rotenone, Valeric acid, RIPA lysis buffer, antibodies against inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2) and glial fibrillary acidic protein (GFAP) were procured from Sigma-Aldrich, St. Louis, MO, USA. Protease and phosphatase inhibitor cocktail were procured from Thermo Scientific, USA. Anti-tyrosine hydrolase (Polyclonal rabbit) antibody was obtained from Merck, Germany. The following antibodies were purchased from Cell Signalling Technology, Beverly, MA, USA: LC3, p62, mTOR, phosphor mTOR and p70S6K. Apoptotic polyclonal markers (Bax and Bcl-2) were obtained from Abcam, USA. Monoclonal mouse anti-α-synuclein antibody was purchased from BD Biosciences, San Jose, CA, USA. Anti- Iba-1 antibody was purchased from Wako chemicals, Richmond, VA, USA. Fluorescent secondary antibodies (Alexa Flour 488) were purchased from Thermo Fischer Scientific, Waltham, MA, USA. Biotinylated goat anti-rabbit secondary antibody was purchased from Jackson Immunoresearch, West grove, PA, USA. Biochemical assays were performed using commercially available kits. All other chemicals used in this experiments were provided by local commercial sources (analytical grade quality).

To analyze the neuroprotective effect of Val, we used an established chronic rotenone paradigm which represents one of the most stable toxin based PD models. Rotenone (2.5 mg/kg) preparation were described in detail previously [46]. Pharmacological effects of Val in vivo were examined using the following treatment groups (n = 15). Group I (control) received intraperitoneal injection of myglol and olive oil which are vehicles for rotenone and Val respectively which served as control group. Group II (rotenone) received intraperitoneal injection of rotenone (2.5 mg/kg) once daily for 4 weeks and served as experimental PD model. Group III (rotenone+ Val): Immediately before treatment, Val (40 mg/kg, i.p) was prepared and administered once daily for four weeks, 30 min prior to ROT administration. Dosage fixed for Val was based on previous study [40]. Group IV received only intraperitoneal injection of Val (40 mg/kg, i.p) once daily for four weeks and served as drug control. Body weight of experimental animals were obtained every 5 days for a total period of 4 weeks.

At the end of the experiments, animals were anesthetized using pentobarbital (40 mg/kg body weight) and perfused via intracardial infusion with 0.01 M phosphate-buffered saline at pH 7.4. For immunohistochemical studies, after infusion with 0.01 M PBS, animals were again perfused with 4% paraformaldehyde for whole body fixation. Brains were then removed from the skull and post-fixed again in 4% paraformaldehyde for 48 h at 4 °C followed by cryoprotection using 30% sucrose solution for three consecutive days (4 °C). For biochemical studies, midbrain and striatum were dissected on dry ice and immediately homogenized using KCl buffer (Tris–HCl 10 mM, NaCl 140 mM, KCl 300 mM, ethylenediaminetetraacetic acid 1 mM, Triton-X 100 0.5%) at pH 8.0 supplemented with protease and phosphatase inhibitor. The homogenates were centrifuged at 14,000× g for 20 min (4 °C) and the supernatant was used for evaluation of lipid peroxidation, antioxidant enzymes and proinflammatory cytokines using spectrophotometric measurements and enzyme-linked immunosorbent assays (ELISA).

This assay was performed to assess the extent of lipid peroxidation in experimental animals using North West Life Science (Vancouver, WA, USA) MDA detection kit. Briefly, 250 μL of samples or calibrators were incubated with thiobarbituric acid followed by rigorous vortexing. After 1 h incubation at 60 °C, the mixture were centrifuged at 10,000× g for 2–3 min and the reaction mixture was transferred to cuvette. Spectra was measured at 532 nm and the results were expressed as μm MDA/mg protein.

Levels of reduced glutathione in tissue homogenates were measured as previously described [46] using Sigma’s glutathione assay kit (Sigma-Aldrich Chemie GmbH, Steinheim). In brief, samples were first deproteinized with 5% 5-sulfosalicylic acid solution, centrifuged to remove the precipitated protein and supernatant was used to estimate GSH. Ten microlitre samples or standards were incubated with 150 μL of working mixture (assay buffer +5,5′-dithiobis (2-nitrobenzoic acid) + GSH reductase) in 96-well plates for 5 min. Diluted NADPH solution (50 µl) was added into each well and mixed thoroughly. Absorbance was measured at 412 nm with the kinetics for 5 min by using the microplate reader. Results were expressed as μm GSH/mg protein.

Cayman assay kits (Cayman Chemicals Company, Ann Arbor, MI, USA) were used to assess antioxidant enzyme [superoxide dismutase (SOD) and catalase (CAT)] activities in experimental animals. Catalase assay: Twenty microliter of samples or standards and 30 μL of methanol was added to the assay buffer (100 μL) in 96-well plates. To this mixture, twenty microliter of hydrogen peroxide was added and incubated for 20 min at room temperature (RT) to initiate the reaction. Following incubation, 30 μL of Potassium hydroxide was used to terminate the reaction, followed by subsequent addition of catalase purpald (30 μL) and catalase potassium periodate (10 μL). The plate was incubated for 5 min at room temperature in a shaker and the plate was read at 540 nm using microplate reader. Catalase activity was expressed as nmol/min/mg protein. Superoxide dismutase assay: Ten microliters of samples or standards were added in 96-well plates. Twenty microliter of xanthine oxidase was added to initiate the reaction. The reaction mixture was mixed for few seconds and incubated (covered) for 30 min at RT. Absorbance was read at 450 nm using microplate reader. The activity of SOD was expressed as units/mg protein.

Nitrite levels in experimental animals were assessed using commercially available R&D Nitrite kit (Minneapolis, MN, USA). Briefly, nitrite standard or samples (50 µL) were added to 50 µL of reaction diluent in a 96-well plate (supplied along with the kit). Fifty microliters of Griess reagent was then added into each well and the reaction mixture in the plate was mixed by gentle tapping. The plate was incubated at room temperature for ten minutes and absorbance was read at 540 nm. Nitrite levels were expressed as µmol/mg protein.

To analyze the effect of Val on rotenone induced pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and MMP-9, we used commercially available ELISA kits (BioSource International Inc., Camarillo, CA, USA). Briefly, 96 well plates were coated with 100 μL of capture antibody (diluted) and incubated overnight at room temperature. Following incubation, each well was washed using wash buffer (0.05% Tween 20 in PBS 0.01 M pH 7.4) and blocked with blocking buffer [1% bovine serum albumin in PBS (300 μL)] for 1 h. The plates were then washed with washing buffer and standards or samples (100 μL) were added into each well and incubated for 2 h at RT. Hundred microliter of detection antibody was then added into each well and the plate was incubated at room temperature for 2 h. Following incubation, the plates were washed and 100 μL of working solution (1:200, streptavidin horseradish peroxidase) was added and the plate was incubated for 20 min. The wells were then exchanged with 100 μL substrate solution and the plate was again incubated further for 20 min. Finally, stop solution [2N H₂SO₄, (50 μL)] was added into each well and the contents in the plate were mixed plate by gentle tapping. The plate was read immediately at 450 nm using microplate reader. The results were expressed as pg/mg protein.

Immunofluorescence staining was performed to analyze the anti-inflammatory effect of Val in experimental animals. Twenty micrometer thick striatum sections were washed twice with PBS and incubated for 1 h with blocking reagent (10% normal goat serum in PBS 0.3% Triton-X 100) at room temperature. The sections were then incubated with anti-rabbit Iba-1 (1:1000) and anti-rabbit GFAP (1:1000) antibodies for overnight at 4 °C. After incubation, the sections were washed twice with PBS and incubated with corresponding fluorescent secondary antibody (Alexa 488 anti-rabbit) for 1 h at RT. The stained sections were again washed twice with PBS and mounted using Vectastain fluorescent mounting media (with DAPI). The images were taken under Nikon Eclipse Ni fluorescent microscope.

A minimum of three coronal sections of the similar level of striatum from each brain were used to analyze activated astrocyte and microglia. Activated astrocytes and microglia were assessed using previously published method [79]. Three different fields of equal area were randomly chosen and analyzed using the Image J software (NIH, Bethesda, MD, USA). Briefly, an outline was drawn around the region of interest and area, circularity, mean fluorescence was measured, along with several adjacent background readings. The total corrected cellular fluorescence (TCCF) was calculated using the formula, TCCF = integrated density–(area of selected cell × mean fluorescence of background readings). All readings were measured by an observer blind to the treatment conditions to guarantee the lack of bias. Results were represented as percentage of control.

The expression of α-synuclein, COX-2, iNOS, Bax, Bcl-2, mTOR, phospho mTOR, p62, LC3 and p70S6K in striatum of experimental animals were assessed using previously published western blotting protocol [46]. In brief, tissues were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitor and centrifuged at 12,000 rpm for 20 min. Equal amount of protein (20 µg) from each sample were loaded and separated on SDS-PAGE. The separated proteins were then electrotransferred onto a PVDF membrane by semi-dry transfer method (BIO-RAD). After blocking (1 h in 5% nonfat dry milk in TBS at RT), the membranes were incubated with α-synuclein (1:750), COX-2 (1:2000), iNOS (1:1000), Bax (1:2000), Bcl-2 (1:500), mTOR (1:1500), phospho mTOR (1:900), p62 (1:900), LC3 (1:800) and p70S6K (1:900) overnight at 4 °C. After incubation, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes are then washed and the bands were visualized using Enhanced Chemiluminescence Pico Kit (Thermo Fisher Scientific). The blots were stripped and re-probed for β-actin (1:5,000; monoclonal mouse; EMD Millipore, Billerica, MA, USA) as a loading control and densitometry analysis was done using “Image J” analysis software.

Cryoprotected animal brains were serially sectioned (20 µm) using a cryostat (Leica, Wetzlar, Germany) as described previously and the sections were washed twice with 0.01 M of PBS, pH 7.4. The sections were incubated for no longer than 10 min with 1% hydrogen peroxidase (in PBS) to inactivate tissue peroxidase. After two washes with PBS, the sections were blocked using blocking reagent (10% normal goat serum in PBS containing 0.3% Triton-X 100) for 30 min at RT and incubated with goat anti-rabbit tyrosine polyclonal antibody (1:1000) overnight at 4 °C. Sections were then rinsed twice with PBS and incubated with biotinylated secondary anti-rabbit (1:1000) antibody for 1 h at room temperature. After incubation, sections were developed using the avidin-biotin peroxidase complex system (ABC kit, Vectastain, CA, USA) followed by 3,3′ diaminobenzidine (DAB) to visualize and analyze TH immunoreactivity. Stained sections were then coverslipped using DPX mounting medium and the slides were viewed under a light microscope (Olympus, Hamburg, Germany).

To analyze the loss of TH immunoreactive (TH-ir) neurons in the SNc area, three different levels of the medial terminal nucleus region were counted, and the average value was represented as percentage of control [46]. Striatal fiber loss was analyzed by measuring the optical density of TH-ir dopaminergic fibers in the striatum using Image J software. The optical density of TH-ir fibers at three different fields from each section with equal area within the striatum was measured for each rat, and an average of three areas were calculated and depicted as percentage of control. The optical density of the overlying cortex was taken as background measure and subtracted from the value generated from the striatum. The counting of TH-ir neurons and optical density of the TH-ir fibers were carried out by an investigator blind to the experimental groups.

Protein concentration from each sample was quantified using Pierce BCA protein assay kit (Thermo Fisher Scientific) following the manufacturer’s instruction.

Data were expressed as the mean value ± SEM. One-way analysis of variance followed by Tukey’s test was performed to calculate the statistical significance between various groups using SPSS 12 software. In all the experiments, p < 0.05 was considered statistically significant.