Vitamin D attenuated 6-OHDA-induced behavioral deficits, dopamine dysmetabolism, oxidative stress and neuro-inflammation in mice.

Abstract

Background:

L-DOPA, the predominant therapy for Parkinson’s disease (PD) is associated with motor deficits after prolonged use. The nigrostriatal tract, a primary target of neurodegeneration in PD, contains abundant Vitamin-D receptors, suggesting a potential role for VD in the disease. Therefore, we tested the impact of Vitamin D₃ (V₃) in a mouse model of PD.

Methods:

PD was induced in adult male C57BL6 mice by a single intrastriatal injection of 6-hydroxydopamine. Two weeks post lesion, these mice received injections of a vehicle, VD₃, L-DOPA, or a combination of VD₃/L-DOPA and compared with sham controls. Treatment lasted three weeks, during which motor-cognitive neurobehaviour was assessed. Five weeks post lesion, brains were collected and striatal levels of the following proteins assessed: tyrosine hydroxylase (TH), dopamine decarboxylase (DDC), monoamine oxidase (MAO-B), Catechol-O-methyl transferase (COMT), dopamine transporter (DAT), brain-derived neurotrophic factor (BDNF), microglia marker (CD11b), inflammation (IL-1β), apoptotic signaling (BAX) and oxidative stress (p47phox).

Results:

Treatment with VD₃ attenuated behavioural deficits induced by 6-OHDA, protein associated with dopamine metabolism and biomarkers of oxidative stress. VD₃ significantly increased contralateral wall touches, exploratory motor and cognitive activities. VD₃ significantly enhanced the expression of TH, DAT, BDNF, while significantly reducing expression of MAO-B, CD11b, IL-I β and p47phox.

Conclusion:

VD₃ reversed some of the 6-OHDA induced changes in proteins involved in modulating the dopamine system, behavioural deficits and oxidative stress biomarkers. The data suggests that VD₃ might be beneficial in reducing L-DOPA dosage, thereby reducing problems associated with dosage and prolonged use of L-DOPA in PD management.

BACKGROUND

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. It is clinically characterized by tremors, rigidity, slowness of movement, and postural imbalance. The disease is likely a result of combinations of genetic and environmental factors [1, 2]. Among environmental factors, evidence indicates that vitamin D may be implicated in the development of PD [3]. Vitamin D is a steroid hormone with pivotal roles in a variety of organs, including the brain [4, 5]. It is obtained from the diet and can be made in the skin from sunshine exposure.

Evidences support an association between the deficiency of Vitamin D and PD in human [6–8]. Although these have been severely criticized, and therefore controversial. Several clinical studies have shown that patients with PD have significantly lower circulating concentrations of 25-hydroxyvitamin D (25-OHD), a clinically relevant and stable biomarker of vitamin D status, compared with healthy controls [9, 10]. In addition, vitamin D supplementation and working outdoors have been observed to be inversely associated with PD [11]. On the contrary, there are reports that show no association between 25-OHD concentration and PD risk [12].

The dopaminergic synapse is the principal site of action of current PD treatments [13], by increasing dopamine synthesis (L-DOPA treatment), inhibiting dopamine catabolism (COMT or MAO inhibition) or by directly activating postsynaptic dopamine receptors [14]. These approaches boost the activity of the dopaminergic synapses. Hence, the dopaminergic synapse is an interesting target for new therapeutic alternatives in the management of PD. L-DOPA is associated with L-DOPA-induced dyskinesia following its prolonged usage [15]. Furthermore, L-DOPA therapy has been reported to be ineffective in attenuating cognitive impairment, a non-motor deficit associated with PD which is quiet debilitating and reduces the quality of life in PD patients [16].

6-hydroxydopamine (6-OHDA) is one of the most extensively used neurotoxin for inducing PD in rodents. Though the 6-OHDA-induced model of PD does not mimic all the classical features observed in PD patients, but it reliably and consistently reproduces the main cellular processes involved in PD, such as neurodegeneration, oxidative stress, neuro-inflammation, and neuronal death. Similarly, the 6-OHDA model does not reproduce the pathological presence of protein aggregate termed Lewy bodies [17]. Understanding the factors and mechanism that can render the dopaminergic neurons vulnerable to degeneration, as well as the relationships between neurodegeneration, microglia activation, neuro-inflammation and apoptosis in the 6-OHDA model is fundamental to identifying potential therapeutic targets for PD [18].

6-OHDA lesions are commonly induced unilaterally, as bilateral dopamine denervation is associated with aphagia, adipsia, and weight loss [19]. Also, unilateral intrastriatal injections with 6-OHDA causes progressive loss of dopaminergic neurons, simulating nigrostriatal damage [20]. At different levels of the nigrostriatal tract, injection of 6-OHDA causes different degrees of DA degeneration [21] and these have different impacts behaviourally and histochemically. Striatal Injection of 6-OHDA causes a graded and progressive loss of substantia nigra neurons [22], which is useful as a partial DA depletion model in studies of functional recovery [21]. It therefore mimics the earlier stage of the disorder [23].

Receptors involved in the control of calcium might be potential alternative targets for relieving the synaptic denervation and associated degenerative changes in the brain caused by L-DOPA [24]. In this regard, Vitamin D receptor (VDR) is a steroid receptor widely distributed in the brain and highly concentrated in the nigrostriatal tract and motor cortex [25]. Vitamin D (VD) and its receptor are involved in numerous brain processes, including neurotrophism, neuro-immunomodulation, inflammation, neuroplasticity, neurotransmission, cytoskeletal stability and regulation of neuronal calcium signalling [26].

Moreover, VD reportedly affects the production of amino acid neurotransmitters. Altered GABA levels are found in the brains of rodents fed with a VD-deficient diet [8] and a significant reduction of glutamate decarboxylase GAD-67 and GAD-65 protein levels has been observed in adult VD-deficient mice. Developmental vitamin D deficiency has been linked to significant disturbances in dopamine neurotransmission in rats [7]. Although evidence points to the ability of VD to modulate neurotransmission [27], as far as we know, there is dearth of data providing a systematic evaluation of the effects of VD on dopamine synthesis, catabolism and transport.

Therefore, to elucidate the role of VD in dopamine neurotransmission, we examined the expression of key enzymes involved in dopamine neurotransmission: tyrosine hydroxylase (TH), dopamine decarboxylase (DDC), monoamine oxidase-B (MAO-B), cathecol-O-methyl transferase (COMT) and dopamine transporter (DAT). We also investigated the impact of VD₃ administered alone or in combination with L-DOPA on motor-cognitive deficit in 6-OHDA lesioned mice.

Biological processes such as oxidative stress [28], microglia activation [29], inflammation [30], cell death [18], are culprit in the pathophysiology of PD [31]. VD₃ has been reported to be involved in these processes both in normal and disease conditions. Therefore, the effects of VD₃ on the biomarkers of the aforementioned processes in mice model of PD was investigated.

MATERIALS AND METHODS

Drugs

All the drugs used were of analytical grade. 6-OHDA hydrobromide and pargyline hydrochloride was a product from Sigma Aldrich. L-DOPA and Benserazide were obtained from TCI, America while Apomorphine, Desipramine hydrochloride and Cholecalciferol (VD₃) were products of US Pharmacopeia.

Mice and surgical induction of Parkinson’s disease

Age-matched (13 weeks), adult male C57BL6 mice with an average weight of 27.5 g were used in this study. The mice were procured from the Jackson laboratory (Bar Harbour, ME). All studies were performed according to NIH guidelines for animal care and use and as approved by the Institutional Animal Care and Use Committee of the two collaborating institutions with approval numbers: (16–015) and (UERC/ASN/2017/738). Mice were housed at a maximum of five per cage, under 12-h light:12-h dark conditions with ad libitum access to food and water.

Experimental Parkinsonism was achieved by slight modification of the method previously described [32]. Briefly, vehicle (saline 0.9% containing 0.02% ascorbic acid) or 3μg of 6-OHDA was unilaterally injected once in the right striatum. Thirty minutes prior to the surgery, desipramine hydrochloride (25mg/kg) and pargyline hydrochloride (5mg/kg) were administered intraperitoneally (i.p.). Desipramine and pargyline were administered prior to injection of 6-hydroxydopamine to increase the selectivity (of dopaminergic neurons) and efficacy of 6-OHDA-induced lesions. Twenty minutes after the administration of the desipramine and pargyline solution, mouse was anaesthetized with ketamine/xylazine (100/20mg/kg i.p.), and observed for absence of withdrawal and blink reflexes.

The fur on the middle top of the mouse’s head was shaved. Topical analgesic (lidocaine) and antibiotics were applied on the scalp. The area of the scalp to be worked upon was sterilized with betadine solution. 6-OHDA was wrapped with aluminium foil and kept on ice all through the procedure.

The mouse was carefully fixed on the stereotaxic frame by inserting warmed incisor and ear bars into the mouth and ears respectively, such that the head was completely flat and could not be moved in either direction. To ensure this, the incisor bar was placed at a level relative to the ear bars.

Using a scapel, 1cm incision was made through the scalp along the midline of the head, the skin was retracted to expose the skull, and then dried using gauze. Using striatal co-ordinates of: anterior posterior: +1.1 mm, mediolateral: +1.5 mm relative to bregma, a mark was made at this coordinate and a drill bit was used to bore a hole (1mm in diameter) through the skull at the point marked. Bone fragments were carefully removed to reveal the surface of the brain. The Hamilton syringe was loaded with the 6-OHDA solution and gently lowered into the brain to reach a depth of 3.0 mm (dorsoventral co-ordinate). A final volume of 2μl of vehicle or 6-OHDA was injected once into the right striatum of mice at the rate of 0.5μl/min. The syringe was kept in place for about five minutes to allow 6-OHDA diffuse away from the injection site. It was then slowly retracted over a period of 3minutes, pausing at intervals to prevent the back flow of the toxin. The scalp was sutured, followed by topical application of antibiotics and analgesic. The mouse was removed from the stereotaxic frame, placed in a warm cage and closely monitored till recovery. Adequate post-surgical care was adopted to avoid dehydration and reduce morbidity.

Animal screening, selection criteria and Parkinsonian assessment

Fifty-four mice went through 6-OHDA lesion. Two weeks after lesion, mice were subjected to cylinder (a non-drug based) and apomorphine (drug-based) behavioural tests for estimating the extent and success of lesion. Mice that showed a contralateral deficit of less than 45% in the cylinder test were included in further experiments. In addition, mice were administered 0.1 mg/kg apomorphine i.p. to determine rotational response to the dopamine agonist. Only mice that displayed contralateral rotation greater than 50 turns per 30 min were included in further experiments. Out of fifty-four 6-OHDA-lesioned mice (which were initially not randomized into groups), 40 passed the inclusion criteria and were divided randomly into four test groups of 10 mice each (untreated 6-OHDA, VD₃, L-DOPA and VD₃/L-DOPA groups). Another group of 10 mice which went through surgery but not lesioned constituted the sham group, totalling 50 mice (divided into five groups).

Animal grouping/experimental schedule

Two weeks after surgical lesions, 6-OHDA-lesioned mice were treated with either VD₃ (30 mg/kg s.c.), L-DOPA (10 mg/kg, i.p.) or a combination of VD₃/L-DOPA daily for 21 days while the PD group was left untreated. The sham control group received the vehicle (5 ml/kg i.p.). L-DOPA was co-administered with Benserazide (7.5 mg/kg i.p.) to inhibit its peripheral decarboxylation thereby enhancing its central bioavailability. To ensure that the experiment was well-controlled, all the mice were subjected to the same conditions and received the same number of injections, both i.p. and s.c. but different treatments. During behavioural assessments on day 8 and 16, batteries of tests (cylinder, horizontal bar, open field and novel object recognition tests) for motor and cognitive performances were conducted thirty minutes after administration of treatment/intervention. On the 35th day post lesion (last day of treatment), the mice were euthanized, brains collected and the striatum was processed for quantitative expression of TH, MAO-B, COMT, DDC, DAT, brain-derived neurotrophic factor (BDNF), BAX (Bcl-2-associated × protein), CD11b, interleukin-1β (IL-1β), p47phox via western blot analysis

Behavioural studies

From the pool of 10 mice in each group, 5–6 mice per group were randomly picked for behavioural assessment. Behavioural studies were performed during the day between 9:00 and 15:00 hr. The testing was randomized per group and each mouse was tested twice (on day 8 and 16).

Cylinder test:

This was assessed using a modified version of a test described previously [33]. Mice were put individually in a transparent Plexiglas cylinder (15 cm diameter, 35 cm height) and video recorded. No habituation to the cylinder prior to the test was allowed. The number of wall touches (contacts with fully extended digits) executed independently with the ipsilateral and the contralateral forepaw were recorded for 10 mins. Simultaneous paw touches were excluded from the analysis. Data was expressed as a percentage of contralateral paw touches calculated as: (contralateral)/(ipsilateral + contralateral) paw touches × 100.

Horizontal bar test:

Using a modified method [34], a horizontal bar of 38 cm long, 3cm in diameter, held 49 cm above the bench surface by a wooden support column at each end was used. Each mouse was held by the tail, quickly slanted backward about 20 cm and rapidly raised and allowed to grasp the center point of the horizontal bar with its forepaws only, while the tail was released and simultaneously starting the stop watch. The time taken by each mouse to reach either end of the bar was recorded. The cut-off time was 30 sec.

Open field test (OFT):

This test was used to measure the exploratory and locomotive behaviour of the mice. A wooden cage with an open field area was marked to contain 15 × 15 squares of 18 cm × 18 cm each. The activities of each mouse on the open field were recorded for the duration of 10 mins using a high definition video recorder kept at a safe distance to cover the entire field. The number of lines crossed (horizontal exploration), rearing frequency (vertical exploration) and freezing time (motor block) by each mouse was later scored by independent investigators. Olfactory cues were reduced by removing mouse litter and cleaning the test field with ethanol.

Novel Object Recognition (NOR):

This test was carried out to assess non-spatial short term/working memory in animals as described previously [35]. A 45 cm × 50 cm opaque box was used. Three days prior to the test, the mice were exposed to the box to familiarize them with the environment for 10 mins each day (habituation session). On the 4th day, mice were allowed to explore two identical objects placed 5 cm apart from each other and from the walls of the box for 10 mins (Trial 1; T1) (training section). An inter-trial time (resting phase) of about 60 mins was observed after which each mouse was returned to the testing field for the second trial (T2). Time allowed for T2 was also 10 mins during which one of the old objects was replaced with a novel object (test session). Exploration was scored when the nose or vibrissae of the mouse was about 2 cm from the object, while sitting on the object was excluded. A mouse not used in the experiment was allowed to explore the arena prior to habituation, training, and testing so that the field would have a familiar odour to the first mouse each day. The location of the objects was changed frequently while the box arena and objects used were thoroughly wiped with 70% ethanol after each test before introducing the next mouse in order to reduce olfactory cues [35]. The session was video recorded and analysed off-line. Object T1 was different from T2 (in shape and texture). The same objects were used on day 8 and 16. The time spent on exploring the old object in T1 and new object in T2 was used to estimate the memory index calculated as: [Time spent exploring new object/total time spent exploring both objects] × 100.

Western blotting and protein quantification

The lysate (20 μl) containing 20 μg of protein was resolved on SDS-PAGE electrophoresis. After subsequent transblotting (wet transfer), Polyvinylidene fluoride membrane (PVDF) was incubated in Tris-buffered saline (with 0.01% Tween 20) (TBST) for 15 minutes at room temperature. Thereafter, the membrane was blocked in 3% Bovine serum albumin (prepared in TBST) for 50 mins at room temperature. The protein of interest, and control (GAPDH) were detected using the following primary antibodies; Mouse anti- TH (Cell Signaling; Danvers, MA, USA), Rabbit anti- BDNF (PeproTECH; New Jersey, USA), Rabbit anti- MAO-B (Abcam; Cambridge, UK), Rabbit anti- DDC (Cell Signaling), Rabbit anti- DAT (abcam), Rabbit anti-GAPDH (Cell Signaling), Rabbit anti- COMT (Proteintech, Illinois, USA), Rabbit anti-BAX (Cell Signaling), Mouse anti- CD11b (Abcam), Rabbit anti- IL-1β (Cell Signaling) and Rabbit anti- p47phox (Enzo life Sci; New York, USA). All primary antibodies were diluted in the blocking solution at 1: 500–1,000. The primary antibodies were detected using HRP-conjugated secondary antibodies (goat anti-rabbit and goat anti-mouse; Invitrogen (California, USA); dilution of 1: 5,000–10,000). The membrane was scanned using Chemidoc scanner (Bio-Rad laboratories; California, USA) to reveal the protein bands, which were quantified using Bio-Rad’s image lab software.

Statistical analysis

Values obtained from the experiments were reported as Mean ± SEM and analysed using Graph Pad Prism (Version 6.0). Differences between groups were determined by one-way ANOVA followed by Tukey test for post-hoc comparisons. The level of significance was considered at p<0.05.

RESULTS

VD₃ and VD₃/L-DOPA enhance motor-cognitive behavioural indices in 6-OHDA-induced mice

In the cylinder test, 6-OHDA induced significant deficit (p<0.05) in the spontaneous asymmetrical use of contralateral paw. VD₃ or L-DOPA (p<0.05) and VD₃/L-DOPA (p<0.01) reversed the deficit compared to PD group. Also, there was a significant increase (p<0.05) in the use of contralateral paw in the VD₃/L-DOPA group compared with L-DOPA mice. VD₃, L-DOPA or the combination attenuated the 6-OHDA induced deficits in contralateral paw usage (Fig. 2A).

Figure 2:

VD₃ attenuated 6-OHDA-induced deficits in motor and cognitive behaviour. Treatment with VD₃ decreased the percentage spontaneous use of contralateral forepaw in the cylinder test (A); reduced the time spent on bar in the horizontal bar test on day 16 (C) but not on day 8 (B); improved exploratory motor activity as depicted by the number of lines crossed in the open field test on days 8 (D) and 16 (E); increased rearing behaviour on days 8 (F) and 16 (G); improved cognitive impairment as shown by increased memory index in NOR test on day 16 (I) but not on day 8 (H); and failed to improve freezing time (J). C; control, PD; Parkinson’s disease, VD₃; vitamin D₃, DOPA; L-DOPA, VD₃/DOPA; combination of VD₃ and L-DOPA. Data are presented in mean ± SEM of 5–6 mice. Panel A: F (4, 27) = 11.24, p < 0.0001. Panel B: F (4, 24) =6.924, p= 0.001. Panel C: F (4, 24) = 50.10, p < 0.0001. Panel D: F (4, 29) = 18.18, p< 0.0001. Panel E: F (4, 29) = 27.64, p< 0.0001. Panel F: F (4, 24) = 6.279, p= 0.0019. Panel G: F (4, 24) = 9.653, p= 0.0002. Panel H: F (4, 27) = 5.578, p= 0.0027. Panel I: F (4, 29) = 8.068, p= 0.0003. Panel J: F (4, 24) = 3.830, p= 0.0181. *p<0.05, **p<0.01 vs control; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs PD; ^βp<0.05 vs DOPA; ^αp<0.05 vs VD₃.

In the horizontal bar test, (Figs. 2B & C) 6-OHDA induced a significant (p<0.01) increase in the time taken for mice to move through the bar when compared with control group on days 8 & 16, signifying a decline in motor performance. L-DOPA (p<0.05) and VD₃/L-DOPA (p<0.01) groups showed significant decrease in the time spent on bar when compared with the PD group. On day 16 (Fig. 2C), interventions with VD₃, L-DOPA and VD₃/DOPA significantly improved 6-OHDA induced deficit in motor performance.

In the open field assessment of exploratory motor activity (Fig. 2D), on day 8, interventions with VD₃ and VD₃/L-DOPA (p<0.01) significantly increased the number of lines crossed following 6-OHDA lesion. VD₃/L-DOPA group showed an increase in exploratory activity compared with L-DOPA group (p<0.05). Similar result was observed on day 16 (Fig. 2E), however L-DOPA significantly (p<0.01) increased the exploratory activity compared with PD. VD₃, L-DOPA or the combination attenuated the 6-OHDA induced deficits in exploratory activity.

Assessment of the rearing behaviour (vertical exploratory activity) as depicted in (Figs. 2F & G) shows that 6-OHDA caused a significant reduction in rearing (p< 0.01). Intervention with VD₃ significantly (p<0.05) improved the rearing behaviour compared with untreated PD mice. On day 16, (Fig. 2G), treatment with VD₃ (p<0.01), L-DOPA (p<0.05), and VD₃/L-DOPA (p<0.001) significantly improved the 6-OHDA-induced deficit in rearing behaviour.

Lesion with 6-OHDA induced cognitive deficit depicted by low memory index (MI) on day 8 (p<0.05, Fig. 2H) & day 16 (p<0.01, Fig. 2I). Treatment with VD₃ improved the 6-OHDA induced cognitive deficit (p<0.01) on day 16, but not on day 8 suggesting a time-dependent effect. On day 16 (Fig. 2I), VD₃/L-DOPA treatment showed a significant (p<0.01) improvement in 6-OHDA induced cognitive deficit.

Striatal 6-OHDA lesion led to an increase in time spent by mouse without engaging in any physical activity (motor block freezing time) (p<0.05). All the treatment groups did not significantly attenuate the freezing time (Fig. 2J).

VD₃ or VD₃/L-DOPA improves dopamine metabolism and transport in 6-OHDA-induced mice

The effects of 6-OHDA-induced dopaminergic neurodegeneration and VD₃ administration on proteins (TH, DDC, COMT, MAO-B, DAT), involved in dopamine neurotransmission and transport were assessed. There was a significant decrease in striatal expression of TH following 6-OHDA lesion (p<0.01). Treatment with VD₃, L-DOPA & VD₃/L-DOPA (p<0.01 & p<0.001 respectively) reversed the 6-OHDA induced decrease in TH expression. Also, the VD₃/L-DOPA group had a significant increase in TH expression (p<0.05) compared with VD₃ mice (Fig 3A).

Figure 3:

VD₃ altered the expression of proteins involved in dopamine metabolism and transport following striatal injection of 6-OHDA. Intervention with VD₃ increased the expression of TH (B); had no effect on the expression of DDC except when combined with L-DOPA (increase) (D); decreased the expression of MAO-B (F); no significant effect on the expression of COMT (H) and increased the expression of DAT (J). Representative blots showing the expression of TH (A), DDC (C), MAO-B (E), COMT (G) and DAT (I) in the striatum. The band densities of blots were quantified respectively (B, D, F, H and J). GAPDH was used as internal control. TH; tyrosine hydroxylase, DDC; dopamine decarboxylase, MAO-B; monoamine oxidase-B, COMT; cathecol-O-methyl transferase, DAT; dopamine transporter. Panel B: F (4, 9) = 51.51, p= 0.0003. Panel D: F (4, 9) = 16.79, p= 0.0042. Panel F: F (4, 9) = 20.45, p= 0.0027. Panel H: F (4, 9) = 12.58, p= 0.008. Panel J: F (4, 9) = 28.84, p= 0.0012. *p<0.05, **p<0.01 vs control; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs PD; ^βp<0.05 vs DOPA; ^αp<0.05 vs VD₃.

DDC was significantly expressed in mice treated with L-DOPA (p<0.05) & VD₃/L-DOPA (p<0.01), but not with VD₃ when compared with PD group. VD₃/L-DOPA (p<0.05) significantly increased the expression of DDC compared to control mice (Fig 3C).

MAO and COMT are responsible for enzymatic degradation of dopamine at the synapse. There was elevated expression of striatal MAO-B following 6-OHDA lesion. Administration of VD₃, VD₃/L-DOPA (p<0.01) & L-DOPA (p<0.05) reversed the 6-OHDA induced increase in MAO-B expression (Fig. 3E).

Lesion with 6-OHDA caused increase in the expression of COMT (p<0.05). Treatment with VD₃/L-DOPA significantly (p<0.01) reduced the expression of COMT following 6-OHDA lesion (Fig. 3G).

There was a significant reduction in the expression of DAT following 6-OHDA lesion (p<0.01). Treatments with VD₃ (P<0.05), VD₃/L-DOPA and L-DOPA (P<0.01) reversed the 6-OHDA induced decrease in DAT expression (Fig. 3I).

VD₃ or VD₃/L-DOPA increases BDNF expression but reduces oxidative stress, microglial, inflammation and apoptotic signalling in 6-OHDA-induced mice.

The expression of BDNF was significantly reduced following 6-OHDA lesion (p<0.01). Administration of VD₃ (p<0.01) and VD₃/L-DOPA (p<0.001) reversed the 6-OHDA induced decrease in the expression of BDNF. Its expression was elevated in the VD₃/L-DOPA group compared with L-DOPA mice (p<0.01) (Fig. 4A).

Figure 4:

VD₃ altered the expression of markers of neurotrophic factor, oxidative stress, microglial, inflammation and apoptotic signalling following striatal injection of 6-OHDA. Administration of VD₃ increased the expression of BDNF (B); decreased the expression of CD11b (D), p47phox (F), and IL-1β (H); had no effect on the expression of BAX but reduced it when combined with L-DOPA (J). Representative blots showing the expression of BDNF (A), CD11b (C), p47phox (E), IL-1β (G) and BAX (I) in the striatum. The band densities of blots were quantified respectively (B, D, F, H and J). GAPDH was used as internal control. BDNF; brain-derived neurotrophic factor, p47phox; phagocyte NADPH oxidase organizer, BAX; Bcl-2-associated × protein, CD11b; marker of microglial, IL-1β; interleukin-1β. Panel B: F (4, 9) = 61.20, p= 0.0002. Panel D: F (4, 9) = 7.415, p= 0.0248. Panel F: F (4, 9) = 11.27, p= 0.0095. Panel H: F (4, 9) = 22.58, p= 0.0017. Panel J: F (4, 9) = 8.003, p= 0.0212. *p<0.05, **p<0.01 vs control; ^#p<0.05, ^##p<0.01, ^###p<0.001 vs PD; ^αp<0.05 vs VD₃.

Similarly, 6-OHDA induced a significant (p<0.01) increase in the expression of CD11b. Treatments with VD₃ and VD₃/L-DOPA significantly (p<0.05) reduced the expression of CD11b following 6-OHDA lesion (Fig. 4C).

p47phox is a primary regulator of NADPH oxidase activity and was found to be significantly (p<0.05) increased as a result of 6-OHDA lesion. This effect was reversed by treatment with VD₃ (p<0.05) (Figs 4E).

Lesion with 6-OHDA caused increase in the expression of IL-1β (p<0.01). Administration of VD₃ or VD₃/L-DOPA (p<0.01) reversed the 6-OHDA induced increase in the expression of IL-1β. In addition, L-DOPA group showed increased expression of IL-1β compared with control mice (p<0.05) (Fig. 4G).

6-OHDA induced lesion caused an increase in the expression of BAX (p<0.05). Only the VD₃/L-DOPA group showed a significant decrease in the expression of BAX (p<0.05) following 6-OHDA lesion (Fig. 4I).

DISCUSSION

PD is a motor dysfunction disorder (accompanied by some non-motor defects) resulting from degeneration of dopaminergic neurons in the substantia nigra pars compacta and consequently, depletion of dopamine in the striatum. Hence, therapeutic interventions that could delay or protect these neurons from neurodegeneration might be helpful. The observed decline in motor function in the PD-induced mice. was evident by an increase in the time spent on the horizontal bar, a reduction in the use of contralateral forelimbs as shown in the cylinder test and a decrease in exploratory motor activity in the open field test.

Activation of VDR via its stimulation by VD treatment either alone or in combination with L-DOPA (additively) reversed this motor-cognitive impairment. VD₃ improved the observed motor decline by causing a decrease in the time taken to walk on the horizontal bar, an increase in the use of contralateral forelimbs in the cylinder test and improving the exploratory motor activity in the open field test (Fig 2 A–G). This corroborates the report of Wang et al. [2001], wherein rats pre-treated with calcitriol prior to 6-OHDA-induced medial fore-bundle lesions had greater locomotor activity than saline treated rats [36]. Given the roles of dopamine in movement and behaviour, compounds that can augment dopamine release may promote the restoration of dopaminergic function and consequently improve motor function. VDR knockout (VDR-KO) mice showed impaired motor function evident in the swim test, and reduced duration of mobility when compared to control. The behavioural characterization of VDR-KO mice revealed changes consistent with diminished muscular development and motor impairment characterized by reduced stride length and habituation in the open field test [37], morphological brain changes and behavioural impairments. Moreover, some of the clinical presentations of PD, such as falls, fractures, and balance problems, have been attributed to low levels of circulating VD. Similarly, VD therapy reportedly improved rigidity, akinesia and also decrease L-DOPA dosage in some PD patients [1].

In this study, desipramine and pargyline were administered prior to injection of 6-hydroxydopamine to increase the selectivity (of dopaminergic neurons) and efficacy of 6-OHDA-induced lesions respectively [32]. There is evidence that some anti-depressant treatment can increase the functional output of dopaminergic system. For example, chronic administration of tricyclic antidepressant drug such as desipramine enhances the locomotor stimulant effect of amphetamine sub sensitivity of dopamine auto receptors and super sensitivity of postsynaptic dopamine auto receptors [38].

One of the reliable non-drug behavioural tests employed in screening potential molecules for the management of PD is cylinder test, which assesses the spontaneous forelimb lateralization, taking advantage of the natural exploratory instinct of rodents to a new environment. It has been postulated that the higher the percentage contralateral uses of forepaw during drug screening with cylinder test, the more likely its effectiveness in PD treatment. In this study, intervention with VD₃ increased the percentage contralateral use of forelimb thus making it a potential candidate.

The reason for this neuroprotective effects of VD₃ is more likely to be due to its ability to rescue or further protect other dopaminergic neurons from the cytotoxic effect of 6-OHDA which in turn promote the striatal release of dopamine. This is also supported by the fact that VD induces expressions of TH, neurotrophins, and reduces oxidative stress, microglial activation and apoptosis [6], thus promoting the conversion of tyrosine to dopamine. Modulation of these factors and biological processes by VD₃ has profound protective properties on the well-being of dopaminergic neurons thereby improving the symptoms and slowing down the progression of PD.

Apart from motor symptoms characterized in PD, a wide range of non-motor deficits can also appear in the course of the disease; including cognitive impairment. In the present study, memory index (MI) was low in PD mice. MI, an index of cognitive function based on the novel object recognition test, is a behavioural test for assessing non-spatial, cortical and hippocampal-dependent learning and memory task. In mice lesioned with the 6-OHDA, there was increase in the time spent on the familial object than the new object resulting in a low MI which translates to cognitive impairment. From our data, VD₃ improved cognitive decline by increasing the MI which was depicted by improvement in the time spent by the mice on new object compared with the old object. In line with our finding, rats supplemented with VD reportedly performed better in the Morris water maze test, a hippocampal-dependent learning and memory task, while the withdrawal of VD from the animals’ diet worsened cognitive impairment. VD deficiency during foetal development resulted in memory and learning impairment. Similarly, several epidemiological studies have shown correlation between low VD concentrations and increased risk of cognitive decline and Alzheimer’s disease (AD) [39]. It is plausible that the mechanism through which VD₃ mediated the observed improvement in motor functions and reduction in cognitive impairment is by decreasing the expression of NADPH oxidase-mediated release of ROS (as depicted by p47phox), thus acting against oxidative stress, inflammatory activity and microglial activation. All of which are central to neuronal oxidative damage, a key player in the progression of PD.

To elucidate the role of VD₃ on dopaminergic neurotransmission, enzymes and transporter involved in dopamine metabolism were examined. We confirmed that treatment with VD₃ after striatal 6-OHDA lesion significantly attenuated neurotoxin-induced decrease in striatal TH protein level (Fig 3A), thus an upregulation of TH expression. It has been shown that VD increases expression of TH, the rate limiting enzyme in dopamine synthesis by increasing the expression of genes encoding it [27], thereby enhancing the evoked release of dopamine and thus, providing significant neuroprotection against degeneration of dopaminergic neuron in rats treated with neurotoxin.

DDC, an enzyme which converts L-DOPA to dopamine, was highly expressed after VD₃ intervention more than in control and PD mice (though in combination with L-DOPA) thus, enhancing dopamine release which in turn, is suggestive of anti-Parkinsonian action. Though there are scarce data on the effects of VD₃ on DDC enzyme and dopamine release in neurotoxin–induced model of PD in rodents. Nevertheless, the observed VD₃-induced increase in the expression of DDC in the lesioned striatum of 6-OHDA treated mice is similar to the mechanism underlying the use of amantadine as an anti-parkinsonian drug in combination with L-DOPA, wherein it acts by increasing DDC activity. Similarly, Arai et al. [2003] also related the anti-parkinsonian effect of amantadine to its dopaminergic action, particularly, in its ability to potentiate L-DOPA-induced elevation of striatal dopamine release [40]. Therefore, VD₃ might have possibly acted especially when co-administered with L-DOPA by enhancing dopamine neurotransmission (though not measured in this study). This may also explain the gross improvement in motor neurobehavioral decline observed in 6-OHDA-lesioned mice treated with VD₃ or VD₃/L-DOPA (Figs. 2A–D).

The expressions of MAO-B and COMT, the catabolic enzymes of dopamine, were dampened in PD mice treated with VD₃ or VD₃/L-DOPA. Following dopaminergic neurotransmission, MAO and COMT enzymes act by inactivating dopamine into its metabolites: 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The ability of VD₃ to inhibit MAO-B and COMT expression (Figs. 2E & G) is in line with the mechanism of action of some drugs used as adjunct with L-DOPA in the treatment of PD. Such drugs as MAO-B inhibitors (Rasagiline) or COMT-inhibitors (Entacapone) prevent dopamine metabolism thereby enhancing the preservation of striatal dopamine concentration, though these drugs are not without their unwanted side effects. Hence, VD₃ could have acted by reducing or inhibiting the activities of these catabolic enzymes, which in turn is thought to prevents or slows down the degradation of dopamine consequently, promoting the availability of dopamine for neuro-transmission. A contrary report to our finding showed that chronic administration of calcitriol in rats induced upregulation of tyrosine hydroxylase, without a change in dopamine status. The report showed a significant increase in the concentrations of dopamine metabolites (DOPAC and HVA) and enhanced MAO and COMT expression [27]. The discrepancies in these results could be attributed to the research methodology. For instance, the authors did not lesion any part of the brain to model PD. Apart from administration of calcitriol, the rats were fed with a formularized 1000 IU VD rodent diet, difference in animal used; Sprague-Dawley rats Vs C57BL6 mice, and the form of VD administered. However, a measure of the levels of dopamine and its metabolites, (not determined in this study) along with the activities of these metabolic enzymes will likely provide a better understanding of dopamine neurotransmission, thus, a limitation of the present study.

Enzymatic degradation does not completely account for inactivation of dopamine. After the action of dopamine at the synapse, DAT clears it off by active reuptake into the presynaptic vesicles for subsequent release. The observed reduction in the expression of DAT infers that clearance of dopamine might be reduced. The unrecycled dopamine is capable of undergoing auto-oxidation thereby increasing toxicity in PD. Also, dopamine is not repackaged for future release hence another probable reason for dopamine shortage in the PD mice but this was attenuated by VD which increased DAT expression and was thought to increased dopamine availability. Neurotrophic factors are important for neuronal survival following injury. These include BDNF, glial cell-derived neurotrophic factor (GDNF) and cerebral dopamine neurotrophic factor (CDNF). Of importance, is BDNF with its receptors well expressed in the striatum and SN of the brain [41]. VD₃ intervention restored the decreased BDNF expression in PD-mice. VD₃ is a potent inducer of GDNF in rat’s glioma cells. Also, the brains of pups from VD deficient dams are characterized by diminished expression of BDNF. Furthermore, VD was reported to protect against dopaminergic neuronal loss from 6-OHDA lesioned rats by increasing GDNF and partially restores TH expression in SN and striatum [6]. Similarly, we observed an increase in TH and BDNF expressions in mice treated with VD₃ after striatal injection of 6-OHDA. Therefore, we infer that VD₃ protects against dopamine neurotoxicity induced by 6-OHDA by restoring TH in the striatum and inducing BDNF which blocked 6-OHDA-induced dopaminergic neuronal loss.

The interplay between inflammation, microglial activation and oxidative stress is central in understanding the mechanism underlying the pathophysiology of PD. This prompted us to check the effects of VD₃ on these processes. In this study, there was increase in the expressions of IL-1β (pro-inflammatory cytokine), CD11b (marker of microglia) and p47phox (marker and organizer of NADPH oxidase, which in turns drives ROS via super oxide production) [42] in PD mice. We showed that 6-OHDA induces microglia by upregulating the expression of CD11b. Activated microglia secretes not only various neurotoxic molecules but also express different proteins and surface markers. Among different surface markers, CD11b is quite important having immense biological implication. Similarly, it has been documented that in various neuro-inflammatory diseases, the increased CD11b expression corresponds to the severity of microglia activation [43].

The progressive nature of PD involves chronic inflammation-induced neurodegeneration of dopaminergic neurons. Levels of pro inflammatory cytokines, including TNF-α. IL-1β, IL-6 as well as ROS are elevated in the brains of PD patients. . In PD patients, the presence of nitrite and increased expression of inducible nitric oxide (iNOS) in the CSF and Substantia Nigra (SN) respectively [44], reportedly justifies the susceptibility of the SN to oxidative stress [45]. From the on-going, it suggests that oxidative stress through generation of reactive oxygen species and activation of microglia play a cardinal role in the development of PD [18]. As such, targeting these may proffer therapeutic breakthrough in the management of PD. VD₃ protected dopaminergic neurons from inflammatory-mediated neurotoxicity by diminishing the expression of IL-1β, thereby acting as an anti-inflammatory agent. Studies have suggested that inhibiting various inflammatory mediators may be effective in dealing with the progression of many neurodegenerative diseases since neuro-inflammation is central to the mechanism underlying them.

Similarly, VD may inhibit ROS elicited by 6-OHDA by decreasing the expression of p47phox, the main regulator of NADH oxidase which consequently drives ROS production. Knock-out and genetic inactivation of NADPH oxidase exerts a neuroprotective effect and reduced detrimental aspect of pathology in experimental models of PD. NOX₂ activation which is regulated by p47phox, was reported to potentiate microglia pro-inflammatory phenotype and is observed in response to several toxins associated with Parkinsonism. Hence, it is reasonable to think that VD salvaged the dopaminergic neurons by inhibiting the activities of NADPH oxidase which in turn downregulated the 6-OHDA-induced release of ROS thereby, suppressing ROS-mediated neurotoxicity leading to improved neuronal survival [46].

Conclusion

VD₃ reversed some of the 6-OHDA-induced behavioural decline, changes in proteins involved in the modulation of the dopamine system and biomarkers involved in oxidative stress. While our data do not categorically imply that VD₃ would be successful as an adjunct to L-DOPA and much work is still required, it however shows that VD₃ is a promising candidate in providing neuroprotection in the treatment of PD.

Figure 1:

Experimental design showing the treatment time line and animal grouping. 6-OHDA-induced mice model of Parkinson’s disease; PD, vitamin D₃-treated PD mice; VD₃, L-DOPA-treated PD mice; DOPA, combined VD₃ and DOPA-treated PD mice; VD₃/L-DOPA. 6-hydroxydopamine; 6-OHDA, subcutaneous; s.c., intraperitoneal; i.p., i.s.; intrastriatal, number of days post treatment; D.